Alkenyl Glucosinolates Research Articles

The Influence of Nitrogen and Sulfur Fertilization on Oil Quality and Seed Meal in Different Genotypes of Winter Oilseed Rape (Brassica napus L.)

Adequate nitrogen (N) and sulfur (S) fertilization of oilseed rape crops is necessary to obtain good-quality oil and post-extraction rapeseed meal. The aim of this study was to determine the effect of different doses of N fertilization (100, 160 and 220 kg ha−1) and S (0, 30, 60 and 90 kg ha−1) on the value of seeds of three winter oilseed rape genotypes. Two winter oilseed rape genotypes obtained by mutagenesis (cultivar Polka and breeding genotype PN440) were characterized by changed fatty acid profile. The cultivar Polka, type HO (high oleic), had a high content of oleic acid (C18:1, 78.0%) and the breeding genotype PN440, type HOLL (high-oleic and low-linolenic), had a high content of oleic acid (C18:1, 75.0%) and a low content of linolenic acid (C18:3, 3.0%). We also used the canola type of winter oilseed rape cultivar, Monolit. The analysed winter oilseed rape genotypes responded similarly to the N and S fertilization factors with regard to the content of crude fat and total protein in the seeds and the composition of fatty acids in the oil. N fertilization increased the content of glucosinolates (GLS-alkenyl, indole and total) in seeds, whereas S application decreased the content of saturated fatty acids (stearic acid-C18:0) in oil and increased the content of alkenyl and total glucosinolates (GLSs) in seeds. A significant interaction between N and S was observed for crude-fat and total-protein content. This study suggests that ensuring an adequate supply of both nitrogen and sulfur in the soil is essential for optimizing meal and oil quality in different types of winter oilseed rape cultivars. Proper management of these nutrients can lead to improved oil content and overall crop performance.

Subsequent treatment of leafy vegetables with low doses of UVB-radiation does not provoke cytotoxicity, genotoxicity, or oxidative stress in a human liver cell model

Ultraviolet B (UVB) radiation in low but ecological-relevant doses acts as a regulator in the plant's secondary metabolism. This study investigates the effect of UVB radiation from light-emitting diodes (LEDs) [peak wavelength of (290 ± 2) nm] on the biosynthesis of health-promoting secondary plant metabolites (carotenoids, phenolic compounds, and glucosinolates) of green and red leafy vegetables of Lactuca sativa, Brassica campestris, and Brassica juncea followed by evaluation of potential adverse effects in a human liver cell model.UVB radiation led to a significant increase in individual secondary plant metabolites, especially of phenolic compounds and glucosinolates, e.g. alkenyl glucosinolate content. Kaempferol und quercetin glycoside concentrations were also significantly increased compared to untreated plants.The plant extracts from Lactuca sativa, Brassica campestris, and Brassica juncea were used to assess cytotoxicity (WST-1 assay and trypan blue staining), genotoxicity (Comet assay), and production of reactive oxygen species (EPR) using metabolically competent human-derived HepG2 liver cells. No adverse effects in terms of cytotoxicity, genotoxicity, or oxidative stress were detected in an extract concentration ranging from 3.125 to 100 μg ml−1. Notably, only at very high concentrations were marginal cytostatic effects observed in extracts from UVB-treated as well as untreated plants.In conclusion, the application of UVB radiation from LEDs changes structure-specific health-promoting secondary plant metabolites without damaging the plants. The treatment did not result in adverse effects at the human cell level. Based on these findings, UVB LEDs are a future alternative, promising light source to replace currently commonly used high-pressure sodium lamps in greenhouses.

Identification and Characterization of Three Epithiospecifier Protein Isoforms in Brassica oleracea.

Glucosinolates present in Brassicaceae play a major role in herbivory defense. Upon tissue disruption, glucosinolates come into contact with myrosinase, which initiates their breakdown to biologically active compounds. Among these, the formation of epithionitriles is triggered by the presence of epithiospecifier protein (ESP) and a terminal double bond in the glucosinolate side chain. One ESP gene is characterized in the model plant Arabidopsis thaliana (AtESP; At1g54040.2). However, Brassica species underwent genome triplication since their divergence from the Arabidopsis lineage. This indicates the presence of multiple ESP isoforms in Brassica crops that are currently poorly characterized. We identified three B. oleracea ESPs, specifically BoESP1 (LOC106296341), BoESP2 (LOC106306810), and BoESP3 (LOC106325105) based on in silico genome analysis. Transcript and protein abundance were assessed in shoots and roots of four B. oleracea vegetables, namely broccoli, kohlrabi, white, and red cabbage, because these genotypes showed a differential pattern for the formation of glucosinolate hydrolysis products as well for their ESP activity. BoESP1 and BoESP2 were expressed mainly in shoots, while BoESP3 was abundant in roots. Biochemical characterization of heterologous expressed BoESP isoforms revealed different substrate specificities towards seven glucosinolates: all isoforms showed epithiospecifier activity on alkenyl glucosinolates, but not on non-alkenyl glucosinolates. The pH-value differently affected BoESP activity: while BoESP1 and BoESP2 activities were optimal at pH 6-7, BoESP3 activity remained relatively stable from pH 4 to 7. In order test their potential for the in vivo modification of glucosinolate breakdown, the three isoforms were expressed in A. thaliana Hi-0, which lacks AtESP expression, and analyzed for the effect on their respective hydrolysis products. The BoESPs altered the hydrolysis of allyl glucosinolate in the A. thaliana transformants to release 1-cyano-2,3-epithiopropane and reduced formation of the corresponding 3-butenenitrile and allyl isothiocyanate. Plants expressing BoESP2 showed the highest percentage of released epithionitriles. Given these results, we propose a model for isoform-specific roles of B. oleracea ESPs in glucosinolate breakdown.

Natural Variation of Glucosinolates and Their Breakdown Products in Broccoli (Brassica oleracea var. italica) Seeds.

Seeds of 32 pure lines and 6 commercial broccoli cultivars were used to investigate variation in glucosinolates and their breakdown products. The aliphatic glucosinolate content was 54.5-218.7 μmol/g fresh weight, accounting for >90% of the total glucosinolates. The major glucosinolates found were glucoraphanin and glucoerucin in 27 samples and progoitrin in 7 samples. A gas chromatography-flame ionization detector (GC-FID) method was used to identify glucosinolate breakdown products; nine products were directly determined using standards. Using Arabidopsis thaliana lines myb28myb29 and Landsberg erecta to hydrolyze each reference glucosinolate, seven products were tentatively identified. 4-(Methylsulfinyl)butyl isothiocyanate and 5-(methylsulfinyl)pentanenitrile contents were 2.6-91.1 μmol/g fresh weight and 0-35.4 μmol/g fresh weight, respectively, with epithionitriles being more common than nitriles in accessions rich in alkenyl glucosinolate. Additionally, (S)-5-vinyl-1,3-oxazolidine-2-thione was detected in accessions rich in progoitrin. Specific lines with altered glucosinolate profiles and breakdown products were obtained and discussed according to the putative glucosinolate metabolism pathway.

Estimation of additive and epistatic gene effects for phenotypic and biochemical traits in double hyploid lines of winter rape seed ,(Brassica napus L.)

In this paper 60 doubled haploid lines of winter oilseed rape (Brassica napus L.) were studied. Genetic parameters as additive and epistatic effects were estimated for 24 traits. The results indicate the importance of both these effects for number of branches per plant, number of siliques per plant, linoleic acid, total of glucosinolates, total of alkenyl glucosinolates, gluconapin, glucobrassicanapin, progoitryn, napoleiferin and indolyl in both years of this study. Statistically significant epistatic effect and non-significant additive effect for thousand seed weight means that this trait was determined by genes with small individual effects but strong gene by gene interaction effects. Confounding epistatic effects in models suggested that inheritance of this trait is complex and polygenic.

Drought stress modulates secondary metabolites in Brassica oleracea L. convar. acephala (DC) Alef, var. sabellica L.

Consumer preference today is for the consumption of functional food and the reduction of chemical preservatives. Moreover, the antimicrobial properties and health-promoting qualities of plant secondary metabolites are well known. Due to forecasted climate changes and increasing human population, agricultural practices for saving water have become a concern. In the present study, the physiological responses of curly kale Brassica oleracea L. convar. Acephala (DC) var. sabellica to drought stress and the impact of water limitation on the concentration of selected secondary metabolites were investigated under laboratory-controlled conditions. Results indicated that drought stress increased the content of trans-2-hexenal, phytol and δ-tocopherol, and decreased chlorophyll content. Moreover, drought stress increased antioxidant capacity and the expression of AOP2, a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates, and of three genes - TGG1, TGGE and PEN2 - encoding for myrosinases, the enzymes involved in glucosinolate breakdown. The present study shows that water limitation during the growing phase might be exploited as a sustainable practice for producing curly kale with a high concentration of nutritionally important health-promoting bioactive metabolites. © 2019 Society of Chemical Industry.

Overexpression of the MYB29 transcription factor affects aliphatic glucosinolate synthesis in Brassica oleracea

Key messageOverexpression of BoMYB29 gene up-regulates the aliphatic glucosinolate pathway in Brassica oleracea plants increasing the production of the anti-cancer metabolite glucoraphanin, and the toxic and pungent sinigrin.Isothiocyanates, the bio-active hydrolysis products of glucosinolates, naturally produced by several Brassicaceae species, play an important role in human health and agriculture. This study aims at correlating the content of aliphatic glucosinolates to the expression of genes involved in their synthesis in Brassica oleracea, and perform functional analysis of BoMYB29 gene. To this purpose, three genotypes were used: a sprouting broccoli, a cabbage, and a wild genotype (Winspit), a high glucosinolate containing accession. Winspit showed the highest transcript level of BoMYB28, BoMYB29 and BoAOP2 genes, and BoAOP2 expression was positively correlated with that of the two MYB genes. Further analyses of the aliphatic glucosinolates also showed a positive correlation between the expression of BoAOP2 and the production of sinigrin and gluconapin in Winspit. The Winspit BoMYB29 CDS was cloned and overexpressed in Winspit and in the DH AG1012 line. Overexpressing Winspit plants produced higher quantities of alkenyl glucosinolates, such as sinigrin. Conversely, the DH AG1012 transformants showed a higher production of methylsulphinylalkyl glucosinolates, including glucoraphanin, and, despite an up-regulation of the aliphatic glucosinolate genes, no increase in alkenyl glucosinolates. The latter may be explained by the absence of a functional AOP2 gene in DH AG1012. Nevertheless, an extract of DH AG1012 lines overexpressing BoMYB29 provided a chemoprotective effect on human colon cells. This work exemplifies how the genetic diversity of B. oleracea may be used by breeders to select for higher expression of transcription factors for glucosinolate biosynthesis to improve its natural, health-promoting properties.

Identification Of N-Acetyl-S-(3-Cyano-2-(Methylsulfanyl)Propyl-Cysteine as a Major Human Urine Metabolite from the Epithionitrile 1-Cyano-2,3-Epithiopropane, the Main Glucosinolate Hydrolysis Product from Cabbage.

Brassica vegetables such as cabbage or pak choi contain alkenyl glucosinolates which can release epithionitriles and to a lesser degree isothiocyanates upon enzymatic hydrolysis. Here, for the first time, the metabolism of an epithionitrile was investigated in humans, namely 1-cyano-2,3-epithiopropane (CETP). After consumption of Brassica oleracea var. capitata f. alba and Brassica carinata sprouts, the main urinary metabolite of CETP was identified as N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine using an UHPLC-ESI-QToF-MS approach and synthesis of the metabolite. This urinary epithionitrile metabolite is an S-methylated mercapturic acid. No other metabolites were detected. Then, in a preliminary pilot experiment the excretion kinetics of CETP were investigated in three volunteers. After consumption of a B. carinata sprout preparation containing 50.8 µmol of CETP, urinary N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine concentrations were the highest three hours after consumption, ranging from 23.9 to 37.2 µM, and declined thereafter. Thus, epithionitriles are bioavailable compounds that are metabolized similarly to isothiocyanates by the mercapturic acid pathway. In the future, more epithionitrile metabolites should be identified and the pharmacokinetics of these important class of dietary compounds should be assessed in more detail.

Differences in the enzymatic hydrolysis of glucosinolates increase the defense metabolite diversity in 19 Arabidopsis thaliana accessions

Plants of the order Brassicales produce glucosinolates (GS), a group of secondary metabolites that are part of an elaborate defense system. But it is not the GS itself rather its enzymatic hydrolysis products that cause the bioactive effects protecting the plants against pests and pathogens. Thus the enzymatic hydrolysis and a variety of additional influential factors determine the structural outcome of the GS degradation process. To evaluate the possible diversity of defense metabolites a range of 19 Arabidopsis thaliana accessions were selected showing divergence in their geographical origin, in their phenotype, and in their GS profile. These particular accessions accumulate several alkenyl GS, hydroxyalkyl GS, methylthioalkyl GS, and methylsulfinylalkyl GS in their rosette leaves whereas the indole GS contents are relatively invariant, as analyzed by UHPLC-DAD. After tissue disruption the enzymatic formation of GS hydrolysis products was examined and breakdown products were identified and quantified by GC-MS. Great differences in the amount and structure of volatile enzymatic degradation products could be observed in the different accessions, with strong variation in formation of epithionitriles, nitriles, and isothiocyanates. The occurrence of specific GS hydrolysis products was put in relation to relative gene expression profiles of myrosinases and specifier proteins as measured by RT-qPCR, and in relation to relative protein abundance of epithiospecifier protein. Dependent on the different GS profiles and reliant on degradation protein abundance and composition the ecotypes strongly varied in their ability to form isothiocyanates, nitriles and epithionitriles, thus increasing the plants' equipment of defense metabolites.

Monitoring blackleg (Leptosphaeria spp.) ascospore release timing and quantity enables optimal fungicide application to improved oilseed rape yield and seed quality

Stem canker is a damaging disease of winter oilseed rape, caused by ascomycetous fungi Leptosphaeria maculans and L. biglobosa. The initial source of plant infection is ascospores, formed in pseudothecia on stubble from the previous growing season. The aim of this work was to evaluate the effectiveness of fungicide treatments at different times of application in the autumn on quality and quantity of seed yield, in relation to the concentration of ascospores of L. maculans and L. biglobosa in the air. Field experiments were done in Lower Silesia, the region of intensive oilseed rape cultivation, located in south-west Poland. We have demonstrated that, in case of high disease pressure, fungicide treatment against stem canker was most efficient when done 4–11 days after the highest concentration of pathogen ascospores in air. The seed yield, oil and protein content increased by 17.7–18.8 %, 1.60–1.72 % and 0.8–0.9 % respectively. In low disease pressure the optimal spray time was more flexible, but the highest yield was always following the maximum ascospore release. In some cases it also coincided with the increase of oil, without adverse effect on protein content. Furthermore, it coincided with the increase of indole glucosinolates, such as glucobrassicine and 4-hydroxyglucobrassicine, and – besides one studied year – it was also connected with decreased amounts of alkenyl glucosinolates. Both Leptosphaeria species were present in air and in infected plants during their vegetation. The ratio between both species depended on seasons; L. maculans prevailed on leaves in the autumn, whereas L. biglobosa dominated on stems before harvest.

The Brassica epithionitrile 1-cyano-2,3-epithiopropane triggers cell death in human liver cancer cells in vitro.

Glucosinolates are secondary metabolites present in Brassica vegetables. Alkenyl glucosinolates are enzymatically degraded forming nitriles or isothiocyanates, but in the presence of epithiospecifier protein, epithionitriles are released. However, studies on the occurrence of epithionitriles in Brassica food and knowledge about their biological effects are scarce. Epithionitrile formation from glucosinolates of seven Brassica vegetables was analyzed using GC-MS and HPLC-DAD. Bioactivity of synthetic and plant-derived 1-cyano-2,3-epithiopropane (CETP) - the predominant epithionitrile in Brassica vegetables - in three human hepatocellular carcinoma (HCC) cell lines and primary murine hepatocytes was also evaluated. The majority of the Brassica vegetables were producers of nitriles or epithionitriles as hydrolysis products and not of isothiocyanates. For example, Brussels sprouts and savoy cabbage contained up to 0.8 μmol CETP/g vegetable. Using formazan dye assays, concentrations of 380-1500 nM CETP were observed to inhibit the mitochondrial dehydrogenase activity of human HCC cells without impairment of cell growth. At 100-fold higher CETP concentrations, cell death was observed. Presence of plant matrix increased CETP-based toxicity. These in vitro data provide no indication that epithionitriles will severely affect human health by Brassica consumption. In contrast to isothiocyanates, no evidence of selective toxicity against HCC cells was found.

Coselected genes determine adaptive variation in herbivore resistance throughout the native range of Arabidopsis thaliana

The "mustard oil bomb" is a major defense mechanism in the Brassicaceae, which includes crops such as canola and the model plant Arabidopsis thaliana. These plants produce and store blends of amino acid-derived secondary metabolites called glucosinolates. Upon tissue rupture by natural enemies, the myrosinase enzyme hydrolyses glucosinolates, releasing defense molecules. Brassicaceae display extensive variation in the mixture of glucosinolates that they produce. To investigate the genetics underlying natural variation in glucosinolate profiles, we conducted a large genome-wide association study of 22 methionine-derived glucosinolates using A. thaliana accessions from across Europe. We found that 36% of among accession variation in overall glucosinolate profile was explained by genetic differentiation at only three known loci from the glucosinolate pathway. Glucosinolate-related SNPs were up to 490-fold enriched in the extreme tail of the genome-wide [Formula: see text] scan, indicating strong selection on loci controlling this pathway. Glucosinolate profiles displayed a striking longitudinal gradient with alkenyl and hydroxyalkenyl glucosinolates enriched in the West. We detected a significant contribution of glucosinolate loci toward general herbivore resistance and lifetime fitness in common garden experiments conducted in France, where accessions are enriched in hydroxyalkenyls. In addition to demonstrating the adaptive value of glucosinolate profile variation, we also detected long-distance linkage disequilibrium at two underlying loci, GS-OH and GS-ELONG. Locally cooccurring alleles at these loci display epistatic effects on herbivore resistance and fitness in ecologically realistic conditions. Together, our results suggest that natural selection has favored a locally adaptive configuration of physically unlinked loci in Western Europe.

Development and Validation of High-glucoraphanin Broccoli F1 Hybrids and Parental Lines

Sulforaphane is an anticarcinogenic isothiocyanate derived from 4-methylsulfinylbutyl glucosinolate (glucoraphanin), which is abundant in broccoli (Brassica oleracea var. italica) florets. However, some breakdown products from alkenyl glucosinolates present in many broccoli cultivars, particularly oxazolidine-2-thione hydrolyzed from 2-(R)-hydroxy-3-butenyl glucosinolate (progoitrin), have potentially harmful effects on human and animal health. The main objective of this study was to improve the glucoraphanin concentration in F1 hybrids by cross-breeding with inbred lines and doubled haploids. Glucoraphanin concentrations in 31 of the 61 F1 hybrids were significantly higher (P = 0.05) than that of the commercial cultivar (Youxiu) with the highest concentration of glucoraphanin (4.18 μmol·g−1 dry weight) among eight reference cultivars. Sixteen of the F1 hybrids had glucoraphanin concentrations 3-fold higher than that of ‘Youxiu’. Alkenyl glucosinolates were not detected in the new hybrids as a result of the parents having few of these compounds but were found in five reference cultivars. Most F1 hybrids showed moderate indole glucosinolate concentrations and acceptable commercial traits. IL609 and IL702.2 were determined to be promising parental lines as a result of the high glucoraphanin concentration that they and their offspring contained. The findings also indicated that some F1 hybrids do not show the high-glucoraphanin character of their parents; consequently, evaluation of these F1 hybrids for their glucosinolate content is required for breeding high-glucoraphanin broccoli.

Verticillium suppression is associated with the glucosinolate composition of Arabidopsis thaliana leaves.

The soil-borne fungal pathogen Verticillium longisporum is able to penetrate the root of a number of plant species and spread systemically via the xylem. Fumigation of Verticillium contaminated soil with Brassica green manure is used as an environmentally friendly method for crop protection. Here we present a study focused on the potential role of glucosinolates and their breakdown products of the model plant Arabidopsis thaliana in suppressing growth of V. longisporum. For this purpose we analysed the glucosinolate composition of the leaves and roots of a set of 19 key accessions of A. thaliana. The effect of volatile glucosinolate hydrolysis products on the in vitro growth of the pathogen was tested by exposing the fungus to hydrated lyophilized plant tissue. Volatiles released from leaf tissue were more effective than from root tissue in suppressing mycelial growth of V. longisporum. The accessions varied in their efficacy, with the most effective suppressing mycelial growth by 90%. An analysis of glucosinolate profiles and their enzymatic degradation products revealed a correlation between fungal growth inhibition and the concentration of alkenyl glucosinolates, particularly 2-propenyl (2Prop) glucosinolate, respectively its hydrolysis products. Exposure of the fungus to purified 2Prop glucosinolate revealed that its suppressive activity was correlated with its concentration. Spiking of 2Prop glucosinolate to leaf material of one of the least effective A. thaliana accessions led to fungal growth suppression. It is suggested that much of the inhibitory effect observed for the tested accessions can be explained by the accumulation of 2Prop glucosinolate.

Dealing with double trouble: consequences of single and double herbivory in Brassica juncea

In their natural environment, plants are often attacked simultaneously by many insect species. The specificity of induced plant responses that is reported after single herbivore attacks may be compromised under double herbivory and this may influence later arriving herbivores. The present study focuses on the dynamics of induced plant responses induced by single and double herbivory, and their effects on successive herbivores. Morphological (leaf length, area and trichome density) and chemical changes (leaf alkenyl and indole glucosinolates) in Brassica juncea were evaluated 4, 10, 14 and 20 days after damage by the specialist Plutella xylostella alone, or together with the generalist Spodoptera litura. To assess the biological effect of the plant’s responses, the preference and performance of both herbivores on previously induced plants were measured. We found that alkenyl glucosinolates were induced 20 days after damage by P. xylostella alone, whereas their levels were elevated as early as 4 days after double herbivory. Trichome density was increased in both treatments, but was higher after double herbivory. Interestingly, there was an overall decrease in indole glucosinolates and an increase in leaf size due to damage by P. xylostella, which was not observed during double damage. S. litura preferred and performed better on undamaged plants, whereas P. xylostella preferred damaged plants and performed better on plants damaged 14 and 10 days after single and double herbivory, respectively. Our results suggest that temporal studies involving single versus multiple attacker situations are necessary to comprehend the role of induced plant responses in plant–herbivore interactions.

Impact of hydroxylated and non-hydroxylated aliphatic glucosinolates in Arabidopsis thaliana crosses on plant resistance against a generalist and a specialist herbivore

Glucosinolates (GSs) are part of a two-component defence system, characteristic for the Brassicales, including the model species Arabidopsis thaliana (L.) Heynh. The defence activity of GSs is associated with different side chain structures. The AOP genes are central in side-chain modification. AOP2 mediates formation of alkenyl GS from a methylsulfinyl precursor, whereas AOP3 catalyzes production of hydroxy-alkyl GSs from the same precursor. Although several studies have assessed the role of GSs in plant defence, the function of specific aliphatic GSs in plant defence is still not clarified. Structural different GSs may influence insect herbivores differentially. We created a set of plant lines derived of a cross between two A. thaliana accessions, Gie-0 × Sap-0, which dominantly accumulate either 3-methylsulfinylpropyl GS or 3-hydroxypropyl GS. The generalist Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae) and the crucifer-specialist Pieris brassicae (L.) (Lepidoptera: Pieridae) were used as model insects, to study effects of individual aliphatic GSs on lepidopteran herbivores with a different feeding specialization. However, the experiments revealed that weight gain of S. exigua and P. brassicae third and fourth-larval instars was similar on both chemotypes. But leaf consumption of the generalist was higher on 3-methylsulfinylpropyl-producing lines with low GS levels (23.2 μmol g−1) than on 3-hydroxypropyl-producing lines that contained a more than twofold higher amount of GSs (60 μmol g−1). In contrast, no differential effects of non-hydroxylated and hydroxylated GSs were found on the specialist P. brassicae. Our study indicates that there is no simple relationship between GS content and insect responses.

A heuristic method of searching for interesting markers in terms of quantitative traits

Selection of parental lines is important in plant breeding programmes. Marker-assisted selection is an alternative to classical selection methods, which are expensive and time consuming. Marker-assisted selection aims to find molecular markers that are linked to genes that determine quantitative traits of interest. Classical statistical methods require particular assumptions to be fulfilled, which is difficult to check if the analyses are performed automatically. In this article, we present a heuristic method to find interesting markers for quantitative traits. This method includes various strategies that depend on what makes a genotype interesting to a plant breeder. This approach was applied to eighteen parental lines of winter oilseed rape F1 CMS ogura hybrids with observation of 597 markers. The traits of interest were seed yield and alkenyl glucosinolate content. Fifty-seven markers were selected for further study. The most prominent marker was OPY 02~1830. Marker-assisted selection is the first step of analysis, which can then be followed up by a more formal statistical analysis for a smaller set of interesting markers.

Glucosinolates – Secondary Plant Products as Important Complex Interaction in our Biosphere

Glucosinolates are the substituted esters of thio amino acids and their synthesis is based on the corresponding amino acids. Methionine and cysteine are the natural donors in the case of the Brassica plants and L tryptophane in the indole glucosinolates, respectively. In Brassica genus, alkenyl glucosinolates are mostly present and their content and composition differ as far as the development stage and the part of the plant are concerned. The indole glucosinolates are present in minority. Their role of sulphur supply is questioned by their very low content between 2 % in the beginning of vegetation and 0.1 % in its end. Glucosinolates are discussed mostly from the aspect of their anti-nutrition, anti-microbial, anti-fungicidal, and antibacterial effects and as being natural bio-fumigants. Their decomposition products have the mentioned properties. The products originate through prepared passive protection of the two-component system. From the aspect of these properties, it is useful to divide them into the following three groups according to the characters of their decomposition products. The first group (I), which hydrolyses in the neutral and alkaline environment creates isothio- cyanates. These bio-active compounds form the natural protection of the plant with bio-fumigatory effects, particularly. With their chemical composition they are ranked among natural pesticides with active and passive resistance against diseases and pests, isothiocyanates.Their anti-nutritive effects can be compensated by iodine, contrary to the second group (II). This group is created by hydroxy – glucosinolates, whose decomposition products – iso-thio-cyanates - are not stable and they cycle while producing substituted 2 – oxazolidimethione (goitrine – VTO). These glucosinolates represent a serious problem in feed industry since the VTO has a strong goitrogenic property. The third group (III) – glucosinolates contain the indole group or the benzene ring (Sinalbin), create thio-cyanates during their hydrolysis. The role of indole glucosinolates has not been completely clarified so far. Their anti-carcinogenic effects are studied and they fulfil the role of an active protection. Keywords: Brassica genus, glucosinolates, biosynthesis, hydrolysis, iso-thio-cyanates, physiological function, anti-nutritive effects, natural biocides anti-bacterial, anti-microbial and anti-fungicidal properties, L tryptophane, in-dole glucosinolates, alkenyl glucosinolates, bio-fumigants, 2, –, oxazolidimethione, N, hydroxy-amino acid, aldoxime, hydroxy acid, de-sulpho glucosinolate, Moringaceae, Tovariaceae, Caricaceae, Euphorbiaceae, Salvadoraceae, Limnanthaceae, Tropaelaceae, Bataceae, Gyrostemonaceae, 2-amino-6-methyl-thio-hexane and 2-amino-7-methyl-thio-heptane acids, L-tryptophane, Sinigrin, Gluconapin, Glucobrassicanapin, Progoitrin, Napoleiferin, Sinalbin, Neoglucobrassicin, 4 - Hydroxyglucobrassicin, uridine-biphosphate-glucose, adenosine-phosphosulphate, progoitrine, indole-glucosinolates, parenchymatous tissues, 5-vinyl-2-oxazolidinethione, phyto-alexines, phyto-anticipines, myrosinase represents, bio-active iso-thio-cyanate, Raphanus sativus, Navajo

The characterisation of AOP2: a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates in Arabidopsis thaliana.

BackgroundGlucosinolates, a group of nitrogen and sulfur containing compounds associated with plant-insect interactions, are produced by a number of important Brassicaceae crop species. In Arabidopsis the AOP2 gene plays a role in the secondary modification of aliphatic (methionine-derived) glucosinolates, namely the conversion of methylsulfinylalkyl glucosinolates to form alkenyl glucosinolates, and also influences aliphatic glucosinolate accumulation.ResultsThis study characterises the primary structural variation in the coding sequences of the AOP2 gene and identifies three different AOP2 alleles based on polymorphisms in exon two. To help determine the regulatory mechanisms mediating AOP2 expression amongst accessions, AOP2 5' regulatory regions were also examined however no major differences were identified. Expression of the AOP2 gene was found to be most abundant in leaf and stem tissue and was also found to be light dependent, with a number of light regulatory elements identified in the promoter region of the gene. In addition, a study was undertaken to demonstrate that the Arabidopsis AOP2 gene product is functional in planta. The over-expression of a functional AOP2 allele was found to successfully convert the precursor methylsulfinyl alkyl glucosinolate into the alkenyl form.ConclusionsThe expression of the AOP2 gene has been found to be influenced by light and is most highly expressed in the photosynthetic parts of the Arabidopsis plant. The level of AOP2 transcript decreases rapidly in the absence of light. AOP2 exists as at least three alleles in different Arabidopsis accessions and we have demonstrated that one of these, AOP2-2, is functionally able to convert methylsulfinyl glucosinolates into the alkenyl form. The demonstration of the in planta functionality of the Arabisopsis AOP2 gene is an important step in determining the feasibility of engineering glucosinolate profiles in food plants.

Key Role of Fe2+ in Epithiospecifier Protein Activity

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe(2+) is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe(2+) requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.