The Combination of Selenium and LED Light Quality Affects Growth and Nutritional Properties of Broccoli Sprouts.

Selenium (Se) supplement was combined with different LED light qualities to investigate mutual effects on the growth, nutritional quality, contents of glucosinolates and mineral elements in broccoli sprouts. There were five treatments: CK:1R1B1G, 1R1B1G+Se (100 µmol L-1 Na2SeO3), 1R1B+Se, 1R2B+Se, 2R1B+Se, 60 µmol m-2 s-1 PPFD, 12 h/12 h (light/dark). Sprouts under a combination of selenium and LED light quality treatment exhibited no remarkable change fresh weight, but had a shorter hypocotyl length, lower moisture content and heavier dry weight, especially with 1R2B+Se treatment. The contents of carotenoid, soluble protein, soluble sugar, vitamin C, total flavonoids, total polyphenol and contents of total glucosinolates and organic Se were dramatically improved through the combination of Se and LED light quality. Moreover, heat map and principal component analysis showed that broccoli sprouts under 1R2B+Se treatment had higher nutritional quality and health-promoting compound contents than other treatments. This suggests that the Se supplement under suitable LED lights might be beneficial to selenium-biofortified broccoli sprout production.

Heterotic patterns of primary and secondary metabolites in the oilseed crop Brassica juncea.

Heterosis refers to the superior performance of F1 hybrids over their respective parental inbred lines. Although the genetic and expression basis of heterosis have been previously investigated, the metabolic basis for this phenomenon is poorly understood. In a preliminary morphological study in Brassica juncea, we observed significant heterosis at the 50% flowering stage, wherein both the growth and reproduction of F1 reciprocal hybrids were greater than that of their parents. To identify the possible metabolic causes or consequences of this heterosis, we carried out targeted LC-MS analysis of 48 primary (amino acids and sugars) and secondary metabolites (phytohormones, glucosinolates, flavonoids, and phenolic esters) in five developmental tissues at 50% flowering in hybrids and inbred parents. Principal component analysis (PCA) of metabolites clearly separated inbred lines from their hybrids, particularly in the bud tissues. In general, secondary metabolites displayed more negative heterosis values in comparison to primary metabolites. The tested primary and secondary metabolites displayed both additive and non-additive modes of inheritance in F1 hybrids, wherein the number of metabolites showing an additive mode of inheritance were higher in buds and siliques (52.77-97.14%) compared to leaf tissues (47.37-80%). Partial least regression (PLS) analysis further showed that primary metabolites, in general, displayed higher association with morphological parameters in F1 hybrids. Overall, our results are consistent with a resource-cost model for heterosis in B. juncea, where metabolite allocation in hybrids appears to favor growth, at the expense of secondary metabolism.

Effects of Selenium Supplementation on Glucosinolate Biosynthesis in Broccoli.

Selenium (Se)-enriched broccoli has health-beneficial selenium-containing compounds, but it may contain reduced amounts of chemopreventive glucosinolates. To investigate the basis by which Se treatment influences glucosinolate levels, we treated two broccoli cultivars with 25 µM Na2SeO4. We found that Se supplementation suppressed the accumulation of total glucosinolates, particularly glucoraphanin, the direct precursor of a potent anticancer compound, in broccoli florets and leaves. We showed that the suppression was not associated with plant sulfur nutrition. The levels of the glucosinolate precursors methionine and phenylalanine as well as the expression of genes involved in glucosinolate biosynthesis were greatly decreased following Se supplementation. Comparative proteomic analysis identified proteins in multiple metabolic and cellular processes that were greatly affected and detected an enzyme affecting methionine biosynthesis that was reduced in the Se-biofortified broccoli. These results indicate that Se-conferred glucosinolate reduction is associated with negative effects on precursor amino acid biosynthesis and glucosinolate-biosynthetic-gene expression and provide information for a better understanding of glucosinolate accumulation in response to Se supplementation in broccoli.

UVA, UVB Light, and Methyl Jasmonate, Alone or Combined, Redirect the Biosynthesis of Glucosinolates, Phenolics, Carotenoids, and Chlorophylls in Broccoli Sprouts.

Broccoli sprouts contain health-promoting phytochemicals that can be enhanced by applying ultraviolet light (UV) or phytohormones. The separate and combined effects of methyl jasmonate (MJ), UVA, or UVB lights on glucosinolate, phenolic, carotenoid, and chlorophyll profiles were assessed in broccoli sprouts. Seven-day-old broccoli sprouts were exposed to UVA (9.47 W/m²) or UVB (7.16 W/m²) radiation for 120 min alone or in combination with a 25 µM MJ solution, also applied to sprouts without UV supplementation. UVA + MJ and UVB + MJ treatments increased the total glucosinolate content by ~154% and ~148%, respectively. MJ induced the biosynthesis of indole glucosinolates, especially neoglucobrassicin (~538%), showing a synergistic effect with UVA stress. UVB increased the content of aliphatic and indole glucosinolates, such as glucoraphanin (~78%) and 4-methoxy-glucobrassicin (~177%). UVA increased several phenolics such as gallic acid (~57%) and a kaempferol glucoside (~25.4%). MJ treatment decreased most phenolic levels but greatly induced accumulation of 5-sinapoylquinic acid (~239%). MJ treatments also reduced carotenoid and chlorophyll content, while UVA increased lutein (~23%), chlorophyll b (~31%), neoxanthin (~34%), and chlorophyll a (~67%). Results indicated that UV- and/or MJ-treated broccoli sprouts redirect the carbon flux to the biosynthesis of specific glucosinolates, phenolics, carotenoids, and chlorophylls depending on the type of stress applied.

Selenium treatment differentially affects sulfur metabolism in high and low glucosinolate producing cultivars of broccoli (Brassica oleracea L.).

The effect of selenium (Se) application on the sulfur (S)-rich glucosinolate (GSL)-containing plant, broccoli (Brassica oleracea L. var. italica) was examined with a view to producing germplasm with increased Se and GSL content for human health, and to understanding the influence of Se on the regulation of GSL production. Two cultivars differing in GSL content were compared. Increased Se application resulted in an increase in Se uptake in planta, but no significant change in total S or total GSL content in either cultivar. Also no significant change was observed in the activity of ATP sulfurylase (ATPS, EC 2.7.7.4) or O-acetylserine(thiol) lyase (OASTL, EC 2.5.1.47) with increased Se application. However, in the first investigation of APS kinase (APSK, EC 2.7.1.25) expression in response to Se fertilisation, an increase in transcript abundance of one variant of APS kinase 1 (BoAPSK1A) was observed in both cultivars, and an increase in BoAPSK2 transcript abundance was observed in the low GSL producing cultivar. A mechanism by which increased APSK transcription may provide a means of controlling the content of S-containing compounds, including GSLs, following Se uptake is proposed.

Glucosinolate biosynthesis in Eruca sativa.

Glucosinolates (GSLs) are a highly important group of secondary metabolites in the Caparalles order, both due to their significance in plant-biome interactions and to their chemoprotective properties. This study identified genes involved in all steps of aliphatic and indolic GSL biosynthesis in Eruca sativa, a cultivated plant closely related to Arabidopsis thaliana with agronomic and nutritional value. The impact of nitrogen (N) and sulfur (S) availability on GSL biosynthetic pathways at a transcriptional level, and on the final GSL content of plant leaf and root tissues, was investigated. N and S supply had a significant and interactive effect on the GSL content of leaves, in a structure-specific and tissue-dependent manner; the metabolites levels were significantly correlated with the relative expression of the genes involved in their biosynthesis. A more complex effect was observed in roots, where aliphatic and indolic GSLs and related biosynthetic genes responded differently to the various nutritional treatments suggesting that nitrogen and sulfur availability are important factors that control plant GSL content at a transcriptional level. The biological activity of extracts derived from these plants grown under the specific nutritional schemes was examined. N and S availability were found to significantly affect the cytotoxicity of E. sativa extracts on human cancer cells, supporting the notion that carefully designed nutritional schemes can promote the accumulation of chemoprotective substances in edible plants.

Genomic Prediction of Testcross Performance in Canola (Brassica napus).

Genomic selection (GS) is a modern breeding approach where genome-wide single-nucleotide polymorphism (SNP) marker profiles are simultaneously used to estimate performance of untested genotypes. In this study, the potential of genomic selection methods to predict testcross performance for hybrid canola breeding was applied for various agronomic traits based on genome-wide marker profiles. A total of 475 genetically diverse spring-type canola pollinator lines were genotyped at 24,403 single-copy, genome-wide SNP loci. In parallel, the 950 F1 testcross combinations between the pollinators and two representative testers were evaluated for a number of important agronomic traits including seedling emergence, days to flowering, lodging, oil yield and seed yield along with essential seed quality characters including seed oil content and seed glucosinolate content. A ridge-regression best linear unbiased prediction (RR-BLUP) model was applied in combination with 500 cross-validations for each trait to predict testcross performance, both across the whole population as well as within individual subpopulations or clusters, based solely on SNP profiles. Subpopulations were determined using multidimensional scaling and K-means clustering. Genomic prediction accuracy across the whole population was highest for seed oil content (0.81) followed by oil yield (0.75) and lowest for seedling emergence (0.29). For seed yieId, seed glucosinolate, lodging resistance and days to onset of flowering (DTF), prediction accuracies were 0.45, 0.61, 0.39 and 0.56, respectively. Prediction accuracies could be increased for some traits by treating subpopulations separately; a strategy which only led to moderate improvements for some traits with low heritability, like seedling emergence. No useful or consistent increase in accuracy was obtained by inclusion of a population substructure covariate in the model. Testcross performance prediction using genome-wide SNP markers shows considerable potential for pre-selection of promising hybrid combinations prior to resource-intensive field testing over multiple locations and years.

Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1.

Over the last few decades, research focusing on canola (Brassica napus L.) seed oil content and composition has expanded. Oil production and accumulation are influenced by genes participating in embryo and seed development. The Arabidopsis LEAFY COTYLEDON1 (LEC1) is a well characterized regulator of embryo development that also enhances the expression of genes involved in fatty acid (FA) synthesis. B. napus lines over-expressing or down-regulating BnLEC1 were successfully generated by Agrobacterium-mediated transformation. The constitutive expression of BnLEC1 in B. napus var. Polo, increased seed oil content by 7-16%, while the down-regulation of BnLEC1 in B. napus var. Topas reduced oil content by 9-12%. Experimental manipulation of BnLEC1 caused transcriptional changes in enzymes participating in sucrose metabolism, glycolysis, and FA biosynthesis, suggesting an enhanced carbon flux towards FA biosynthesis in tissues over-expressing BnLEC1. The increase in oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value or glucosinolate (GLS) levels. Suppression of BnLEC1 reduced seed oil accumulation and elevated the level of GLS possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. Collectively, these findings demonstrate that experimental alterations of BnLEC1 expression can be used to influence oil production and quality in B. napus.

Methyl Jasmonate- and Light-Induced Glucosinolate and Anthocyanin Biosynthesis in Radish Seedlings.

Radish sprouts and young seedlings are considered important dietary vegetables in Asian countries. In this study, we investigated the levels of glucosinolate and anthocyanin accumulation in radish seedlings in response to light and methyl jasmonate (MeJA) treatments. MeJA facilitated the accumulation of glucosinolate and anthocyanins under light conditions. The glucosinolate and anthocyanin contents in the radish seedlings that were exposed to light after MeJA treatment were higher than those of the seedlings that were grown in the dark without MeJA. At a concentration of 100 µM, MeJA led to the greatest accumulation of the most glucosinolates under both light and dark conditions. Under light conditions, the levels of glucoraphenin, glucoerucin, and glucotropaeolin accumulation were 1.53-, 1.60-, and 1.30-fold higher, respectively, than those of the control. Remarkable accumulations of glucobrassicin were observed under light conditions (4.4-, 6.7-, and 7.8-fold higher than that of the control following the application of 100, 300, and 500 µM MeJA, respectively). The level of cyanidin in the 300 µM MeJA-treated seedlings was double of that in the control without MeJA treatment. The highest level of pelargonidin was observed after treatment with 500 µM MeJA under light conditions; this level was 1.73 times higher than that in the control. A similar trend of anthocyaninaccumulation was observed in the radish seedlings following MeJA treatment under dark conditions, but the levels of anthocyanins were considerably lower in the seedlings that were grown in the dark. Our findings suggest that light and low concentrations of MeJA enhance the accumulations of glucosinolates and anthocyanins during the development of radish seedlings.

Exogenous methyl jasmonate treatment increases glucosinolate biosynthesis and quinone reductase activity in kale leaf tissue.

Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties 'Dwarf Blue Curled Vates' and 'Red Winter' in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar 'Red Winter' in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001). Concentrations required to double the specific QR activity (CD values) of I3C was calculated at 230 µM, which is considerably weaker at induction than other isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to combined increases in phenolics (quercetin and kaempferol) and GS hydrolysis product concentrations rather than by individual products alone.

Contribution of endogenous plant myrosinase to the antimicrobial activity of deodorized mustard against Escherichia coli O157:H7 in fermented dry sausage.

This work investigated the antimicrobial activity of residual endogenous plant myrosinase in Oriental and yellow mustard powders and a deoiled meal (which contained more glucosinolate than unextracted mustard powder of each type of mustard), against Escherichia coli O15:H7 during dry-fermented sausage ripening. When small amounts of "hot" mustard powder or meal containing endogenous plant myrosinase were added to fully-deodorized powders and a meal of the same type, pathogen reduction rates were enhanced. The higher glucosinolate level in the deoiled mustard meal enabled the use of 50% less mustard in dry sausage to achieve the mandatory ≥5logCFU/g reduction of E. coli O157:H7. The myrosinase-like activity present in E. coli O157:H7 contributed to glucosinolate hydrolysis in sausages with fully-deodorized, deoiled mustard meal, although the period necessary for a 5log pathogen reduction was 14d longer. Yellow mustard derivatives were more potently antimicrobial than Oriental mustard.

Glucosinolate biosynthesis in hairy root cultures of broccoli (Brassica oleracea var. italica).

Here we present previously unreported glucosinolate production by hairy root cultures of broccoli (B. oleracea var. italica). Growth media greatly influenced the growth and glucosinolate content of hairy root cultures of broccoli. Seven glucosinolates, glucoraphanin, gluconapin, glucoerucin, glucobrassicin, 4-methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin, were identified by analysis of the broccoli hairy root cultures. Both half and full strength B5 and SH media enabled the highest accumulation of glucosinolates. In most cases, the levels of glucosinolates were higher in SH and BS media. Among the 7 glucosinolates, the accumulation of neoglucobrassicin was very high, irrespective of growth medium. The neoglucobrassicin content was 7.4-fold higher in SH medium than 1/2 MS, in which its level was the lowest. The 1/2 B5 medium supported the production of the highest amounts of glucobrassicin and 4-methoxyglucobrassicin, the levels for which were 36.2- and 7.9- fold higher, respectively, than their lowest content in 1/2 MS medium. The 1/2 SH medium enabled the highest accumulation of glucoraphanin and gluconapin in the broccoli hairy root cultures, whose levels were 1.8- and 4.6-fold higher, respectively, than their lowest content in 1/2 MS medium. Our results suggest that hairy root cultures of broccoli could be a valuable alternative approach for the production of glucosinolate compounds.

Crambe tataria: the glucosinolate/myrosinase system in vivo and in vitro.

The Crambe tataria glucosinolate/myrosinase system in seeds and leaves of in vivo and in vitro regenerated plantlets, and two callus cell lines was investigated. It was demonstrated that in all the extracts glucosinolates were present and the myrosinase system was operative. There appears to be no discrimination between the glucosinolates used as substrates, but the hydrolysis rates were different regardless of the nature of the side chain. This is one of the first studies demonstrating that undifferentiated cells are able to synthetize glucosinolates and have an operating myrosinase system.

Generation of Se-fortified broccoli as functional food: impact of Se fertilization on S metabolism.

Selenium (Se)-fortified broccoli (Brassica oleracea var. italica) has been proposed as a functional food for cancer prevention, based on its high glucosinolate (GSL) content and capacity for Se accumulation. However, as selenate and sulphate share the initial assimilation route, Se fertilization could interfere with sulphur metabolism and plant growth. Consequently, GSL accumulation could be compromised. To evaluate these potentially adverse effects of Se fertilization, we performed a comprehensive study on sand-grown young broccoli plants (weekly selenate applications of 0.8 µmol plant(-1) via the root) and field-grown adult broccoli plants during head formation (single foliar selenate application: 25.3 or 253 µmol plant(-1) ). The results show that under these conditions, Se application does not affect plant growth, contents of cysteine, glutathione, total GSL, glucoraphanin (major aliphatic GSL) or the expression of BoMYB28 (encoding a functionally confirmed master regulator for aliphatic GSL biosynthesis). Conversely, due to the changed expression of sulphate transporters (BoSULTR1;1, 1;2, 2;1, and 2;2), sulphate and total S contents increased in the shoot of young plants while decreasing in the root. We conclude that broccoli can be fertilized with Se without reduction in GSL content, even with Se accumulation exceeding the level recommended for human consumption.

Production of the cancer-preventive glucoraphanin in tobacco.

Epidemiological studies have demonstrated reduced risk of developing cancer upon consumption of diets rich in cruciferous vegetables. This chemoprevention has been largely attributed to the presence of the natural products glucosinolates, particularly the methionine-derived glucoraphanin from broccoli. Improved nutrition by functional foods or health-promoting dietary supplements is an attractive means for prevention of lifestyle-based diseases. Towards this goal, we have engineered the glucoraphanin pathway into tobacco. First, we engineered elongation of the side chain of methionine to produce the key intermediate dihomo-methionine. This process is catalyzed through two cycles in a chain-elongation pathway that takes place partly in the cytosol and partly in the chloroplast. Second, by coupling the five enzymes of the chain-elongation pathway to eight enzymes of the glucosinolate pathway, we show production of glucoraphanin together with other glucosinolates derived from chain-elongated isoleucine and/or leucine. The conversion of methionine to glucoraphanin is obtained via 14 intermediates. Demonstrating the production of the high-value glucoraphanin in a heterologous host has great potential in the food and medicinal industry as a means to generate a stable, rich source of glucoraphanin for the benefit of human health.

Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the final step in the biosynthesis of the glucosinolate core structure.

The phytotoxin coronatine is a structural analog of octadecanoid signaling molecules, which are well known mediators of plant defense reactions. To isolate novel coronatine-regulated genes from Arabidopsis thaliana, differential mRNA display was performed. Transcript levels of CORI-7 (coronatine induced-7) were rapidly and transiently increased in coronatine-treated plants, and the corresponding cDNA was found to encode the sulfotransferase AtST5a. Likewise, upon wounding, an immediate and transient increase in AtST5a mRNA levels could be observed in both locally wounded and unwounded (systemic) leaves. Furthermore, application of octadecanoids and ethylene as compounds involved in plant wound defense reactions resulted in AtST5a gene activation, whereas pathogen defense-related signals (yeast elicitor and salicylic acid) were inactive. AtST5a and its close homologs AtST5b and AtST5c were purified as His6-tagged proteins from Escherichia coli. The three enzymes were shown to catalyze the final step in the biosynthesis of the glucosinolate (GS) core structure, the sulfation of desulfoglucosinolates (dsGSs). They accept a broad range of dsGSs as substrates. However, in a competitive situation, AtST5a clearly prefers tryptophan- and phenylalanine-derived dsGSs, whereas long chain dsGSs derived from methionine are the preferred substrates of AtST5b and AtST5c. Treatment of Arabidopsis plants with low concentrations of coronatine resulted in an increase in the amounts of specific GSs, primarily glucobrassicin and neoglucobrassicin. Hence, it is suggested that AtST5a is the sulfotransferase responsible for the biosynthesis of tryptophan-derived GSs in vivo.

Production of two volatile glucosinolate hydrolysis compounds in Nasturtium montanum and Cleome chelidonii plant cell cultures.

Callus and suspension cultures established from Nasturtium montanum and Cleome chelidonii were shown to produce glucosinolates by analysis of their hydrolysis products. Large increases in two glucosinolate hydrolysis products were noted when cultures were supplemented with L-cysteine and L-methionine, and further increases were produced in N. montanum with l-tryptophan supplementation.

The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes.

The Arabidopsis ref2 mutant was identified in a screen for plants having altered fluorescence under UV light. Characterization of the ref2 mutants showed that they contained reduced levels of a number of phenylpropanoid pathway-derived products: sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. Surprisingly, positional cloning of the REF2 locus revealed that it encodes CYP83A1, a cytochrome P450 sharing a high degree of similarity to CYP83B1, an enzyme involved in glucosinolate biosynthesis. Upon further investigation, ref2 mutants were found to have reduced levels of all aliphatic glucosinolates and increased levels of indole-derived glucosinolates in their leaves. These results show that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates and suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Capparales, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species.

The presence of CYP79 homologues in glucosinolate-producing plants shows evolutionary conservation of the enzymes in the conversion of amino acid to aldoxime in the biosynthesis of cyanogenic glucosides and glucosinolates.

A cDNA encoding CYP79B1 has been isolated from Sinapis alba. CYP79B1 from S. alba shows 54% sequence identity and 73% similarity to sorghum CYP79A1 and 95% sequence identity to the Arabidopsis T42902, assigned CYP79B2. The high identity and similarity to sorghum CYP79A1, which catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, suggests that CYP79B1 similarly catalyses the conversion of amino acid(s) to aldoxime(s) in the biosynthesis of glucosinolates. Within the highly conserved 'PERF' and the heme-binding region of A-type cytochromes, the CYP79 family has unique substitutions that define the family-specific consensus sequences of FXP(E/D)RH and SFSTG(K/R)RGC(A/I)A, respectively. Sequence analysis of PCR products generated with CYP79B subfamily-specific primers identified CYP79B homologues in Tropaeolum majus, Carica papaya, Arabidopsis, Brassica napus and S. alba. The five glucosinolate-producing plants identified a CYP79B amino acid consensus sequence KPERHLNECSEVTLTENDLRFISFSTGKRGC. The unique substitutions in the 'PERF' and the heme-binding domain and the high sequence identity and similarity of CYP79B1, CYP79B2 and CYP79A1, together with the isolation of CYP79B homologues in the distantly related Tropaeolaceae, Caricaceae and Brassicaceae within the Capparales order, show that the initial part of the biosynthetic pathway of glucosinolates and cyanogenic glucosides is catalysed by evolutionarily conserved cytochromes P450. This confirms that the appearance of glucosinolates in Capparales is based on a cyanogen 'predisposition'. Identification of CYP79 homologues in glucosinolate-producing plants provides an important tool for tissue-specific regulation of the level of glucosinolates to improve nutritional value and pest resistance.

Involvement of cytochrome P450 in oxime production in glucosinolate biosynthesis as demonstrated by an in vitro microsomal enzyme system isolated from jasmonic acid-induced seedlings of Sinapis alba L.

An in vitro enzyme system for the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been established by the combined use of an improved isolation medium and jasmonic acid-induced etiolated seedlings of Sinapis alba L. An 8-fold induction of de novo biosynthesis of the L-tyrosine-derived p-hydroxybenzylglucosinolate was obtained in etiolated S. alba seedlings upon treatment with jasmonic acid. Formation of inhibitory glucosinolate degradation products upon tissue homogenization was prevented by inactivation of myrosinase by addition of 100 mM ascorbic acid to the isolation buffer. The biosynthetically active microsomal enzyme system converted L-tyrosine into p-hydroxyphenylacetaldoxime and the production of oxime was strictly dependent on NADPH. The Km and Vmax values of the enzyme system were 346 microM and 538 pmol per mg of protein per h, respectively. The nature of the enzyme catalyzing the conversion of amino acid to oxime in the biosynthesis of glucosinolates has been subject of much speculation. In the present paper, we demonstrate the involvement of cytochrome P450 by photoreversible inhibition by carbon monoxide. The inhibitory effect of numerous cytochrome P450 inhibitors confirms the involvement of cytochrome P450. This provides experimental documentation of similarity between the enzymes converting amino acids into the corresponding oximes in the biosynthesis of glucosinolates and cyanogenic glycosides.