Factors Causing Variation in Glucosinolates in Oilseed Rape

Milford, G. F. J.; Evans, Eric J.

doi:10.1177/003072709102000107

Cited by 31 publications

(29 citation statements)

References 5 publications

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“…Yearly monitoring of the rapeseed crop (B. napus and B. campestris) in Canada has revealed that above-average levels of GS occur in seed produced during a growing season marked by high temperatures and drought (Canadian Grain Commission). Similar seasonal variations have been observed in Great Britain (Morgan and Cooke, 1989;Milford and Evans, 1991).…”

Section: Enzyme Sourcessupporting

confidence: 81%

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

1994

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Preparing homogeneous UDP-g1ucose:thiohydroximate S-glucosyltransferase (S-CT), the penultimate biosynthetic enzyme of glucosinolates, by standard chromatographic methods has yielded too little protein for adequate purity evaluation, identity verification, and structural analysis. l h e low yields were apparently due to low abundance in source tissues, aggravated by enzyme instability. Here we describe an immunological method for purification of-workable quantities from florets of Brassica oleracea ssp. botrytis (cauliflower). Florets that had undergone browning dueto exposure to sunlight contained higher S-CT activities than are normally found in Brassica tissues. S-CT was adsorbed from crude tissue extracts onto an agarose-monoclonal antibody complex. Elution from the complex required harsh alkaline conditions (pH 11.51, giving extremely variable activity recoveries (maximum 20%). l h e eluate contained two proteins that could be separated readily by preparative polyacrylamide gel electrophoresis or anion-exchange chromatography. The overall S-CT protein recovery was estimated at less than 200 pg/kg of cauliflower tissue. Molecular weight determinations with homogeneous cauliflower S-CT gave relative molecular weight (M,) values of 55,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 57,600 by gel chromatography; isoenzymes with isoelectric point values of 4.80 and 4.95 were identified. A polyclonal antibody raised against denatured enzyme showed broad cross-reactivity in immunoblots with S-CT from a number of Brassica species and other crucifers. l h e monoclonal antibody that was used in the immunopurification was much more specific; it exclusively precipitated S-CT isoenzymes that had their genomic origin in the primary diploids B. oleracea and Brassica campestris. Thus, all of the S-CT was precipitated from the amphidiploid Brassica napus, which is a hybrid of B. oleracea and B. campestris. About half of the S-CT was precipitated from the amphidiploids Brassica carinata and Brassica juncea, which have B. oleracea and B. campestris as one of their parents, respectively. It was shown that the S-CT isoenzymes of B. juncea with M, 55,500 and about 57,000 originate from the parents B. campestris and B. nigra, respectively.GS (sulfated S-glucosyl thiohydroximates) constitute a large group of secondary metabolites occurring in severa1 plant families, including a11 species of the Cruciferae. Their presence in oilseed Brassica crops (Brassica napus and Brassica campestris) has been of great nutritional and therefore economic concern, since the meal fraction is directed to animal feed markets as a protein source. GS have antinutritional properties and cause acute and chronic diseases, particularly * Corresponding author; fax 1-306-975-4839. 425

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Section: Enzyme Sourcessupporting

confidence: 81%

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

1994

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“…For example, per capita consumption in the UK amounts to 45 g/day (13). Milford and Evans reported that the glucosinolate content varied between 9 to 35 µmol/g in crops (14). However the average daily consumption of all glucosinolates in the UK is estimated to be~50 mg (15).…”

Section: Introductionmentioning

confidence: 99%

Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model

Krul¹,

Humblot²,

Philippe³

et al. 2002

Carcinogenesis

147

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Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites.

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“…The authors cast doubt on the involvement of other aldoximeforming activities in glucosinolate biosynthesis. However, it remains an open question whether the Cyt P450 MO activity found in these etiolated seedlings is related to glucosinolate biosynthesis, because previous work indicated that cotyledons do not actively synthesize aromatic or aliphatic/ alkenylglucosinolates (Glen et al, 1990;Bodnaryk, 1991), and glucosinolate biosynthesis in leaf tissues is strongly light dependent (Milford and Evans, 1991). The work on white mustard also showed that this Cyt P450 MO activity was stimulated by jasmonate treatment of the seedlings (Du et al, 1995), although others have found that jasmonates only stimulate indolylglucosinolate formation (Bodnaryk, 1992(Bodnaryk, , 1994Doughty et al, 1995).…”

mentioning

confidence: 99%

Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly)

1997

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B i o c h e m i s t r y and Physiology Department, IACR-Rothamsted, H a r p e n d e n AL5 2JQ, United KingdomOne of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and severa1 very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl-and alkenylglucosinolates are also present i n white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found i n Brassica sp. and are distinct from the tyrosinedependent enzyme i n that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cuz+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation i n either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found i n other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape.Considerable efforts have been made to breed glucosinolates out of the seeds of commercial varieties of canola (Brassica campestris) and oilseed rape (Brassica napus), because the presence of these compounds reduces the nutritional value of rapeseed meal. However, these same secondary metabolites are present in cruciferous vegetables eaten by humans. Their breakdown products (isothiocyanates and others) are the flavor components of mustards, radishes, broccoli, and others. The presence of moderate quantities of such compounds in foodstuffs has been found to have significant beneficia1 effects, such as the stimulation of defensive responses that protect against carcinogens (Jongen, 1996;Rhodes, 1996). The successful reduction in seed glucosinolates in canola and rape is detrimental to the

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Factors Causing Variation in Glucosinolates in Oilseed Rape

Cited by 31 publications

References 5 publications

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic microflora in a dynamic in vitro large-intestinal model

Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly)

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