Genetics of aliphatic glucosinolates. IV. Side-chain modification in Brassica oleracea

The biochemical and genetical relationship between aliphatic glucosinolates which have methylthioalkyl, methylsulphinylalkyl and alkenyl side chains has not been resolved by biochemical studies. In this study, two hypothetical models are tested by the genetic analysis of a backcross population between Brassica drepanensis and B. atlantica. The results support one of the models in which 3-methylthiopropyl glucosinolate is sequentially converted to 3-methylsulphinylpropyl, and then to 2-propenyl glucosinolate, by the action of dominant alleles at two loci. RFLP mapping positioned both loci on the same linkage group homologous to the B. napus N19 linkage group. The implication of the results for the genetic manipulation of glucosinolates in Brassica to improve flavour and nutritional properties, and in order to investigate plant-insect interactions, is discussed.     

Isolation of a chitin-binding lectin, with insecticidal activity in chemically-defined synthetic diets, from two wild brassica species with resistance to cabbage aphidBrevicoryne brassicae

A lectin, with a chitin-binding domain and chitinase activity, is present in significant quantities in the wild brassica speciesB. fruticulosa andB. spinescens but at low levels in cultivated cabbage cv. Offenham Compacta. The lectin, purified >1000 fold after binding to chitin, migrated on SDS-PAGE gels as a single band with a M _r of 14.500. The amino acid composition of the lectin fromB. spinescens indicated high concentrations of asparagine/aspartic acid, glycine, leucine and serine in common with other chitin-binding lectins with insecticidal and antifungal activities. Brassica lectin and the closely related agglutinin from wheatgerm and nettle show significant insecticidal activity when presented toBrevicoryne brassicae in chemically-defined synthetic diets.     

Analysis by DC–EPG of the resistance to Bemisia tabaci on an Mi-tomato line

The tomato Mi gene confers resistance to nematodes, Meloidogyne spp., and to the potato aphid, Macrosiphum euphorbiae (Thomas). Previous greenhouse choice assays with Bemisia tabaci (Gennadius) showed that tomato commercial varieties carrying this gene had significantly lower values of host suitability and whitefly reproduction than varieties lacking Mi. This indicated that Mi, or another gene in its region, could regulate partial resistance. In order to characterise this resistance, probing and feeding behaviour of Bemisia tabaci B-biotype was studied with DC Electrical Penetration Graph (EPG) technique on the near-isogenic tomato lines Moneymaker (without Mi) and Motelle (carrying Mi). Significant differences (P < 0.05) between tomato lines were found in EPG parameters related to epidermis and/or mesophyll tissues. On Motelle, a lower percentage of whiteflies achieved phloem phase and they made more probes before attaining first phloem phase, had a higher ratio (number of probes before first phloem phase)/(total number of probes), had a longer total duration of non-probing time, and a longer time before making the first intracellular puncture and before making the first phloem phase. In contrast, most of the parameters related to phloem phase were found not to differ significantly between these near-isogenic lines. The behavioural data strongly suggest that the partial resistance in the variety Motelle is due to factors in the epidermis and/or mesophyll that inhibit the whiteflies from reaching phloem sieve elements. However, once the stylets reach a sieve element, whitefly behaviour did not differ between the two varieties. Thus, phloem sap of the two varieties appears to be equally acceptable to the whiteflies. Further studies are necessary to provide a better understanding of these mechanisms of resistance to whiteflies in tomatoes.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

Glucosinolates in <Emphasis Type="Italic">Brassica</Emphasis> foods: bioavailability in food and significance for human health

Glucosinolates are sulphur compounds that are prevalent in Brassica genus. This includes crops cultivated as vegetables, spices and sources of oil. Since 1970s glucosinolates and their breakdown products, have been widely studied by their beneficial and prejudicial biological effects on human and animal nutrition. They have also been found to be partly responsible for the characteristic flavor of Brassica vegetables. In recent years, considerable attention has been paid to cancer prevention by means of natural products. The cancer-protective properties of Brassica intake are mediated through glucosinolates. Isothyocianate and indole products formed from glucosinolates may regulate cancer cell development by regulating target enzymes, controlling apoptosis and blocking the cell cycle. Nevertheless, variation in content of both glucosinolates and their bioactive hydrolysis products depends on both genetics and the environment, including crop management practices, harvest and storage, processing and meal preparation. Here, we review the significance of glucosinolates as source of bioactive isothiocyanates for human nutrition and health and the influence of environmental conditions and processing mechanisms on the content of glucosinolate concentration in Brassica vegetables. Currently, this area is only partially understood. Further research is needed to understand the mechanisms by which the environment and processing affect glucosinolates content of Brassica vegetables. This will allow us to know the genetic control of these variables, what will result in the development of high quality Brassica products with a health-promoting activity.     

Leafhoppers on leaves: An analysis of feeding behavior using conditional probabilities

Feeding behavior of four deltocephaline leafhoppers,Graminella nigrifrons, G. oquaka, Amblysellus grex, andDalbulus maidis on maize and johnsongrass was analyzed using an electronic monitoring device. Five distinct waveform patterns were identified: secretion of sheath saliva (salivation), nonvascular probing, nonsieve element ingestion, x-waveform, and phloem ingestion. Waveforms were associated with feeding activities by correlation with light microscopic examination of salivary sheath termination points in leaf tissue and analysis of honeydew excreted by monitored leafhoppers. In previous studies x-waveforms have been reported to occur only when the stylets of homopterans are in contact with the phloem; the function of x-waveforms is poorly understood. There were no differences in time spent salivating or ingesting from nonsieve elements amongG. nigrifrons, G. oquaka andA. grex on either plant.D. maidis differed from other species in phloem probing and feeding behavior; only a small proportion produced x-waveforms, although those that did spent significantly more time in this behavior than other species. Also,D. maidis spent more time than other leafhoppers ingesting from tissues other than sieve elements. Kinetic diagrams of transition probabilities show that probing activities of all species were not random regarding the sequence of behaviors culminating in phloem ingestion. Thirty-five percent ofG. nigrifrons x-waveforms were followed by nonsieve element ingestion. This was consistent with observations showing that salivary sheaths of leafhoppers producing x-waveforms sometimes do not terminate in the phloem, but rather in nearby cells. Phloem ingestion was always preceeded by x-waveforms. The quantitative differences in probing behavior are discussed in relation to ability of these leafhoppers to transmit the phloem-associated maize chlorotic dwarf waikavirus.     

Constitutive and herbivore-inducible glucosinolate concentrations in oilseed rape (Brassica napus) leaves are not affected by Bt Cry1Ac insertion but change under elevated atmospheric CO2 and O3

Glucosinolates are plant secondary compounds involved in direct chemical defence by cruciferous plants against herbivores. The glucosinolate profile can be affected by abiotic and biotic environmental stimuli. We studied changes in glucosinolate patterns in leaves of non-transgenic oilseed rape (Brassica napus ssp. oleifera) under elevated atmospheric CO₂ or ozone (O₃) concentrations and compared them with those from transgenic for herbivore-resistance (Bacillus thuringiensis Cry1Ac endotoxin), to assess herbivory dynamics. Both elevated CO₂ and O₃ levels decreased indolic glucosinolate concentrations in transgenic and non-transgenic lines, whereas O₃ specifically increased the concentration of an aromatic glucosinolate, 2-phenylethylglucosinolate. The herbivore-inducible indolic glucosinolate response was reduced in elevated O₃ whereas elevated CO₂ altered the induction dynamics of indolic and aliphatic glucosinolates. Herbivore-resistant Bt plants experienced minimal leaf damage after target herbivore Plutella xylostella feeding, but exhibited comparatively similar increase in glucosinolate concentrations after herbivory as non-transgenic plants, indicating that the endogenous glucosinolate defence was not severely compromised by transgenic modifications. The observed differences in constitutive and inducible glucosinolate concentrations of oilseed rape under elevated atmospheric CO₂ and O₃ might have implications for plant–herbivore interactions in Brassica crop-ecosystems in future climate scenarios.     

An investigation of the resistance to cabbage aphid in brassica species

Fifteen Brassica species and subspecies, as well as accessions of Arabidopsis thaliana and Eruca sativa, were evaluated for their resistance to the cabbage aphid, Brevicoryne brassicae, in the field and laboratory at Horticulture Research International (HRI) Wellesbourne in 1992. In the laboratory, aphid performance was measured in terms of pre-reproductive period, reproductive period, population increase and insect survival. Using these parameters it was possible to identify brassicas possessing antibiosis resistance. In the field B. brassicae attack was assessed in terms of numbers of insects and it was possible to determine aphid preferences for alighting on different species. High levels of antixenosis and antibiosis resistance were discovered in accessions of Brassica fruticulosa and B. spinescens, in a Brassica juncea breeding line and in Eruca sativa. Partial resistance was found in several other Brassica species. The potential of these various sources as donors of resistance genes to be bred into cultivated brassicas is discussed.     

Tissue localisation of lettuce resistance to the aphidNasonovia ribisnigri using electrical penetration graphs

Electrical penetration graphs (EPG's) ofNasonovia ribisnigri (Mosley) (Homoptera, Aphididae) on resistant and susceptible lettuce (Lactuca sativa, Compositae) showed a large reduction in the duration of the food uptake pattern (E2) on the resistant line. No differences in EPG's were observed before the phloem was reached. Therefore, resistance is believed to be located in the phloem vessel. Both mechanical blocking of the sieve element after puncturing and a difference in composition of the phloem sap are possible resistance factors. However, a chemical factor seems more likely because of the specificity of the resistance againstN. ribisnigri.     

Induced resistance by Myzus persicae in the peach cultivar `Rubira'

The effect of a previous infestation by the green peach aphid Myzus persicae (Sulzer) on the settling behaviour and reproduction of the same aphid species was investigated in the resistant peach cultivar Rubira, and compared with that observed in the susceptible control cultivar GF305. A previous infestation of 48 h triggered induced resistance in Rubira. There were significantly fewer aphids settling on preinfested than on uninfested plants, indicating an increased rejection of Rubira as a host plant. The level of induced resistance in preinfested plants was positively related to the duration of the first infestation. In GF305, previous infestation had no detrimental effect on aphid settlement and even slightly enhanced larviposition by adult females. The aphid probing behaviour after a 48-h preinfestation was also monitored for 8 h with the electrical peneration graph (EPG) technique. On preinfested GF305, most EPG parameters indicated an enhanced host plant acceptance. On preinfested GF305, aphids produced less sieve element salivation and more continuous sap ingestion than on uninfested GF305, indicating that the previous aphids provoked changes in plant properties beneficial to the test aphids. In Rubira, a major induced factor of resistance was thought to be expressed in the sieve element as phloem sap ingestion was 4-fold shorter on preinfested than on uninfested plants. The time taken by the aphid stylets to reach a sieve element was also significantly increased on preinfested Rubira, suggesting the induction of resistance factors outside the phloem. The originality of the Rubira/M. persicae interaction is discussed in the perspective of a better understanding of plant induced responses to aphids.     

Association of gene-linked SSR markers to seed glucosinolate content in oilseed rape (Brassica napus ssp. napus)

Breeding of oilseed rape (Brassica napus ssp. napus) has evoked a strong bottleneck selection towards double-low (00) seed quality with zero erucic acid and low seed glucosinolate content. The resulting reduction of genetic variability in elite 00-quality oilseed rape is particularly relevant with regard to the development of genetically diverse heterotic pools for hybrid breeding. In contrast, B. napus genotypes containing high levels of erucic acid and seed glucosinolates (++ quality) represent a comparatively genetically divergent source of germplasm. Seed glucosinolate content is a complex quantitative trait, however, meaning that the introgression of novel germplasm from this gene pool requires recurrent backcrossing to avoid linkage drag for high glucosinolate content. Molecular markers for key low-glucosinolate alleles could potentially improve the selection process. The aim of this study was to identify potentially gene-linked markers for important seed glucosinolate loci via structure-based allele-trait association studies in genetically diverse B. napus genotypes. The analyses included a set of new simple-sequence repeat (SSR) markers whose orthologs in Arabidopsis thaliana are physically closely linked to promising candidate genes for glucosinolate biosynthesis. We found evidence that four genes involved in the biosynthesis of indole, aliphatic and aromatic glucosinolates might be associated with known quantitative trait loci for total seed glucosinolate content in B. napus. Markers linked to homoeologous loci of these genes in the paleopolyploid B. napus genome were found to be associated with a significant effect on the seed glucosinolate content. This example shows the potential of Arabidopsis-Brassica comparative genome analysis for synteny-based identification of gene-linked SSR markers that can potentially be used in marker-assisted selection for an important trait in oilseed rape. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The influence of glucosinolates and sugars on feeding by the cabbage stem flea beetle,Psylliodes chrysocephala

Agar was used as an artificial substrate to investigate the feeding behaviour of the cabbage stem flea beetle,Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae), an important pest of winter oilseed rape (Brassica napus) in Europe. Both glucosinolates and sugars stimulated feeding when added to agar. The amount of feeding that occurred was affected by the type and concentration of glucosinolate and surgar and also by combinations of components. Although glucosinolates were potent feeding stimulants forP. chrysocephala, they were not a prerequisite for feeding, nor does it seem likely that glucosinolate profiles are used by this species to discriminate amongst cruciferous plants at the gustatory level.     

Glucosinolate polymorphism in wild cabbage (Brassica oleracea) influences the structure of herbivore communities

Natural plant populations often show substantial heritable variation in chemical structure of secondary metabolites. Despite a great deal of evidence from laboratory studies that these chemicals influence herbivore behaviour and life history, there exists little evidence for the structuring of natural herbivore communities according to plant chemical profiles. Brassica oleracea (Brassicaceae) produces aliphatic glucosinolates, which break down into toxins when leaf tissue is damaged. Structural diversity in these glucosinolates is heritable, and varies considerably at two ecological scales in the UK: both within and between populations. We surveyed herbivore attack on plants producing different glucosinolates, using 12 natural B. oleracea populations. In contrast to the results of previous studies in this system, which suffered low statistical power, we found significant differential responses of herbivore species to heritable glucosinolates, both within and between plant populations. We found significant correlations between herbivore infestation rates and the presence or absence of two heritable glucosinolates: sinigrin and progoitrin. There was variation between herbivore species in the direction of response, the ecological scale at which responses were identified, and the correlations for some herbivore species changed at different times of the year. We conclude that variation in plant secondary metabolites can structure the community of herbivores that attack them, and propose that herbivore-mediated differential selection deserves further investigation as a mechanism maintaining the observed diversity of glucosinolates in wild Brassica.     

Histochemical localization of nucleoside triphosphatase activity in assimilate conducting plant tissue

Summary For the histochemical localization of nucleoside triphosphatases at the electron microscopic level, prefixed tissues were incubated with lead nitrate in addition to substrate (GOMORI reaction). While ATP and UTP as substrates gave electron-dense reaction products at the plasmalemma of sieve tubes, companion cells and phloem parenchyma cells, and at plasmodesmata in primary pitfields, AMP gave reaction products only at the tonoplast of parenchyma cells. Since electron-dense deposits also occur in cell walls and vacuoles, energy dispersive X-ray microanalysis was used to distinguish between lead deposits and lead-phosphate deposits. The latter were restricted to the symplast. Among the three plant species used, the leaf bundle phloem ofHordeum distichon showed ATPase activity largely restricted to the phloem cells, except for the thickwalled sieve tubes. Some activity also bordered the chloroplasts of the bundle sheath cells. In the C₄ plantGomphrena globosa, ATPase and UTPase activities appeared to be the greater in phloem parenchyma cells than in sieve tubes. In the phloem of youngMonstera deliciosa roots, ATPase occurred not only at the plasmalemma of sieve tubes, but also around sieve-tube plastids. When compared with AMP as substrate, it appears that nucleoside triphosphates are the natural substrates of the enzyme(s) in the plasmalemma of sieve tubes and phloem parenchyma cells.     

Identification of phloem sieve elements as the site of resistance to silverleaf whitefly in resistant alfalfa genotypes

Experiments were conducted to locate the plant tissue where resistance is expressed against silverleaf whitefly, Bemisia argentifolii Bellows and Perring (Homoptera: Aleyrodidae), in alfalfa, Medicago sativa L. (Fabaceae), genotypes previously shown to have high levels of resistance against this pest. Previous work demonstrated that resistance in the resistant alfalfa genotypes was expressed primarily as high first‐instar mortality; consequently this study focused on first‐instar nymphs. Examination of stylets in cleared leaf tissue indicated that first‐instar nymphs located vascular bundles with equal success on resistant and susceptible alfalfa genotypes. Furthermore, direct current electrical penetration graphs (DC‐EPG) indicated that sieve elements were penetrated and phloem ingestion behavior was initiated with equal success on resistant and susceptible genotypes. Thus, the mechanism of resistance does not reside in tissues encountered by the stylets prior to penetrating a phloem sieve element. Honeydew production (as a proxy for ingestion) was greatly reduced on two resistant genotypes compared to the two susceptible genotypes. The frequency distribution of honeydew production was bimodal, indicating that most individuals on the resistant genotypes produced little or no honeydew while some produced as much honeydew as whiteflies on the susceptible genotypes. This indicates that expression of resistance is an all‐or‐nothing phenomenon; an individual nymph either encounters resistance and cannot sustain ingestion or it does not encounter resistance and ingests just as well as on a susceptible plant. Intermediates are rare. DC‐EPGs indicate that phloem ingestion behavior is significantly reduced on two of the resistant genotypes compared to the susceptible genotypes. The primary reason for this appears to be more frequent termination of phloem ingestion behavior on at least one of the resistant genotypes. On one of the resistant genotypes, the productivity of EPG‐measured phloem ingestion behavior (honeydew produced per min of phloem ingestion behavior) was reduced compared to a susceptible control.     

Electrical penetration graphs of the damson-hop aphid, Phorodon humuli on resistant and susceptible hops (Humulus lupulus)

Electrical penetration graphs (EPG's) were used to locate resistance to Phorodon humuli (Schrank) (Homoptera, Aphididae) in hops (Humulus lupulus, Cannabinaceae). Aphids on those hops showing resistance had a much reduced E2 pattern (uptake of phloem). In addition, many aphids on the resistant plants spent time non-probing within two minutes of withdrawing from the phloem. This was not observed with aphids on susceptible hops. The results suggest that resistance is located in the phloem. The involvement of a mechanical factor such as the blocking of aphid stylets, the presence of antinutritional factors, or simply an inadequate supply of nutrients, are discussed as possible resistance mechanisms.     

Differential abundance of simple repetitive sequences in species ofBrassica and relatedBrassicaceae

SixBrassica species, known as the triangle of U, and four species from related genera were characterized by DNA fingerprinting with simple repetitive oligonucleotide probes. Our results show that CT-, TCC-, and GTG-repeat motifs are equally abundant in the genomes of the sixBrassica species. In contrast, GATA-, GGAT-, and GACA-multimers are unevenly distributed among different species. As judged from the number and strength of hybridization signals, the highest copy number of all three motifs occurs inBrassica nigra, while the lowest is observed inB. oleracea. The abundance of GATA-and GACA-repeats varies in a coordinate way. The amphidiploid genomes ofB. juncea, B. carinata, andB. napus each harbour intermediate amounts of (GATA)₄ and (GACA)₄-detected repeats as compared to their diploid progenitors, thus supporting the concept of the U triangle. GATA-, GACA-, and GGAT-repeats were also abundant inEruca sativa andSinapis arvensis, but not inRaphanus sativus andSinapis alba. These results support the idea thatBrassica nigra is more closely related toSinapis arvensis than to otherBrassica species such asB. rapa andB. oleracea.     

Seed glucosinolates of fourteen wild Brassica species

Seed glucosinolates have been determined for 14 wild Brassica species, by micro-scale GC analysis of silylated derivatives. Of these, 12 were investigated for the first time. The majority of taxa exhibited high alkenylglucosinolate levels, although prop-2-enylglucosinolate appears to be generally absent. Other known methionine-derived glucosinolates predominate in B. tournefortii, B. elongata and B. deflexa. Phenylalanine-derived 4-hydroxybenzylglucosinolate is characteristic of section Brassicaria plants and represents the first finding of this glucosinolate in authenticated Brassica material.     

Penetration of faba bean sieve elements by pea aphid does not trigger forisome dispersal

Immediately after their stylets penetrate a phloem sieve element, aphids inject saliva into the sieve element for approximately 30–60 s before they begin to ingest phloem sap. This salivation period is recorded as waveform E1 in electrical penetration graph (EPG) monitoring of aphid feeding behavior. It has been hypothesized that the function of this initial period of phloem salivation is to reverse or prevent plugging of the sieve element by one of the plant's phloem defenses: formation of P‐protein plugs or callose synthesis in the sieve pores that connect adjacent sieve elements. This hypothesis was tested using the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), and faba bean, Vicia faba L. (Fabaceae), as a model system, and the results do not support the hypothesis. In legumes, such as faba bean, P‐protein plugs in sieve elements are formed by dispersal of proteinaceous bodies called forisomes. Contrary to the hypothesis, the great majority of sieve element penetrations by pea aphid stylets do not trigger forisome dispersal. Thirteen sieve elements were cryofixed early in phloem phase before the aphids could complete their salivation period and the forisomes were not dispersed in any of the 13 samples. However, in these samples, the aphids completed on average a little over half of their normal E1 salivation period before they were cryofixed. Thus, it is possible that sieve element penetration triggered forisome dispersal in these samples but the abbreviated period of salivation was still sufficient to reverse dispersal. To rule out this possibility, 17 sieve elements were cryofixed during R‐pds, which are an EPG waveform associated with sieve element penetration but without the characteristic E1 salivation that occurs during phloem phase. In 16 of the 17 samples, the forisomes were not dispersed. Thus, faba bean sieve elements usually do not form P‐protein plugs in response to penetration by pea aphid stylets. Consequently, the characteristic E1 salivation that occurs at the start of each phloem phase does not seem to be necessary to prevent a plugging response because penetration of sieve elements during R‐pds does not trigger forisome dispersal despite the absence of E1 salivation. Furthermore, as P‐protein plugs do not normally form in response to sieve element penetration, E1 salivation that occurs at the start of each phloem phase is not a response to development of a P‐protein plug. Thus, the E1 salivation period at the beginning of the phloem phase appears to have function(s) unrelated to phloem sealing.