Benzylglucosinolate degradation in heat-treated Lepidium sativum seeds and detection of a thiocyanate-forming factor

Lepidium sativum seeds were dry heated at 125° for varying periods, and also for 30 min at various temperatures. Autolysates were then analysed for benzylglucosinolate degradation products. Whilst heating for 4 hr 20 min at 125° was sufficient to prevent formation of benzyl thiocyanate, just over 7.5 hr at 125° was required before benzyl isothiocyanate also ceased to be produced. This indicates the presence of a discrete, thiocyanate-forming factor in L. sativum seeds, separate from thioglucosidase. After 7.5 hr at 125°, benzyl cyanide continued to be formed, proving that it can be obtained (in relatively small amounts) directly from the glucosinolate even without the influence of any thioglucosidase. In general, isothiocyanate was the more favoured product of glucosinolate degradation following heat treatment of seeds, until the point of thioglucosidase inactivation was approached when nitrile formation took over. It is suggested that the thiocyanate-forming factor is an isomerase causing Z-E isomerization of the glucosinolate aglucone, but that only those glucosinolates capable of forming particularly stable cations are then able to undergo E-aglucone rearrangement to thiocyanate.     

马槟榔种子中的葡糖硫甙硫酸酯和葡糖硫甙酶

自马槟榔(Capparis masaikai L(?)vl.)种子中分离出一种新的葡糖硫甙硫酸酯,即2-羟乙基葡糖硫甙硫酸酯(2-hydroxyethylglucosinolate),得率为干种仁的3％;在葡糖硫甙酶(pH 6.0)作用下,酶解产物为(口恶)唑烷硫酮(oxazolidine-2-thione)。马槟榔种子中的葡糖硫甙酶用1N氯化纳提取,经硫酸铵沉淀,通过葡聚糖G50凝胶过滤层析和羧甲基纤维素离子交换层析,得到纯制品。测得该酶分子量Mr=116 kD,是由二个相近的亚基组成,等电点pI=5.05。与文献中报道从十字花科植物中提取的葡糖硫甙酶是分子量为140 kD的糖蛋白不同,该酶未检出含糖。     

Glucosinolate degradation by Aspergillus clavatus and Fusarium oxysporum in liquid and solid-state fermentation

Two fungal strains, Aspergillus clavatus II-9 and Fusarium oxysporum @ 149, proved to be capable of degrading sinigrin and sinalbin. During the degradation of sinigrin by whole cells of the Aspergillus strain, allylcyanide accumulated in the liquid incubation mixture. After a maximum concentration had been reached, the concentration of allylcyanide decreased as a result of its instability in the medium used. Incubation of cell-free extracts with sinigrin resulted in accumulation of glucose and allylisothiocyanate, suggesting that myrosinase is involved. Experiments with intact cells and cell-free extracts indicate the formation of an as yet unknown intermediate. When sinigrin was degraded by the Aspergillus strain in mustard seed meal under solid-state fermentation (SSF) conditions, no accumulation of allylcyanide or allylisothiocyanate was measured. Degradation of sinigrin by F. oxysporum @ 149 did not result in accumulation of intermediates, neither in liquid incubation mixtures nor in mustard seed meal under SSF conditions. Sinigrin was not degraded during incubation with cell-free extracts of F. oxysporum @ 149. Degradation of sinalbin by A. clavatus and F. oxysporum was measured during fermentation of yellow mustard seed meal under SSF conditions. Both fungi are useful for laboratory-scale SSF of mustard seed meal, thus opening new perspectives for a cost effective detoxification process for raw feed materials. Correspondence to: J. P. Smits     

N+注入诱变选育菜粕硫苷降解菌的研究

为选育能够高效降解菜粕硫苷( Glucosinolates,Gls)的菌株,利用能降解硫苷的益生菌黑曲霉CICC-2238进行N+注入诱变.经过两次N+注入诱变,在诱变能量为20 keV,剂量为60 ×2.5×1013ions/cm2时,筛选到一株高效降解Gls的菌株H-109.与出发菌相比较,其Gls的降解率由28....     

Toxicity of Glucosinolates and Their Enzymatic Decomposition Products to Caenorhabditis elegans

An aquatic 24-hour lethality test using Caenorhabditis elegans was used to assess toxicity of glucosinolates and their enzymatic breakdown products. In the absence of the enzyme thioglucosidase (myrosinase), allyl glucosinolate (sinigrin) was found to be nontoxic at all concentrations tested, while a freeze-dried, dialyzed water extract of Crambe abyssinica containing 26% 2-hydroxyl 3-butenyl glucosinolate (epi-progoitrin) had a 50% lethal concentration (LC₅₀) of 18.5 g/liter. Addition of the enzyme increased the toxicity (LC₅₀ value) of sinigrin to 0.5 g/liter, but the enzyme had no effect on the toxicity of the C. abyssinica extract. Allyl isothiocyanate and allyl cyanide, two possible breakdown products of sinigrin, had an LC₅₀ value of 0.04 g/liter and approximately 3 g/liter, respectively. Liquid chromatographic studies showed that a portion of the sinigrin decomposed into allyl isothiocyanate. The results indicated that allyl isothiocyanate is nearly three orders of magnitude more toxic to C. elegans than the corresponding glncosinolate, suggesting isothiocyanate formation would improve nematode control from application of glucosinolates.     

1982-2012年江苏省审定油菜品种系谱及性状分析

分析了1982-2012年江苏省审定的62个油菜品种的亲本系谱、品种特性以及主要农艺性状和经济性状特点。结果表明,江苏省审定的甘蓝型油菜品种的原始亲本为胜利油菜,低芥酸性状主要源自Zephyr、Primor、Midas和荣山等,低硫苷性状主要源自Tower、Wesroona、Marnoo和Wesbrook等。江苏省审定的油菜品种的主要农艺性状中,生育期逐步缩短,并基本稳定在243 d左右;株高变化较大,因品种而异。4个产量(产油量)构成因素中,含油量年平均增幅最大,尤其是在2003-2012年间,年平均增幅达到0.65%;单株角果数和每角粒数有上升趋势,而千粒重则变化相对较小。受双低性状对产量性状的负效应影响,产量及产油量在1982-2012年间的年平均增幅均不大,但优质常规品种的产量与产油量的年平均增幅较大,分别为23.10 kg/hm2和13.20 kg/hm2。研究结果可为江苏省油菜新品种的选育与推广提供参考。     

Uptake and distribution of sinigrin in microspore derived embryos of Brassica napus L

In Brassica napus, glucosinolates are transported from all parts of the plant into the embryo during seed development. In this study we describe the uptake of the alkenyl glucosinolate sinigrin by microspore derived embryos from high and low glucosinolate genotypes. Microspore derived embryos develop completely isolated from maternal tissues unlike zygotic embryos, which contains glucosinolates transported into the embryo synthesised in the vegetative tissues. The sinigrin in the culture medium was almost completely absorbed by the embryos after three days of culture. The embryos of high and low glucosinolate genotypes were equally capable of absorbing sinigrin from the medium. A significant increase in different alkenyl glucosinolates following feeding of sinigrin suggests induction of biosynthetic enzymes in the embryos. Following excess feeding of sinigrin, we found a strong uptake against a concentration gradient and stable accumulation by the embryos. The glucosinolate was detected in single dissected cotyledons by a photometric test and by HPLC. This test could potentially be useful for screening mutants defective in glucosinolate uptake into the embryo.     

Effects of a Lepidium sativum enzyme preparation on the degradation of glucosinolates

The effects of a crude enzyme extract prepared from Lepidium sativum seeds, on the degradation of three pure glucosinolates (allyl-, benzyl- and 2-phenethyl-) were investigated in the presence of the known enzyme co-factor, ascorbic acid. Isothiocyanates and nitriles were obtained but no thiocyanates. For maximum isothiocyanate formation there was an optimum concentration of ascorbic acid which varied directly with the concentration of substrate but was independent of the particular glucosinolate. Formation of isothiocyanate from any glucosinolate was linear with time but enzymic production of 2-phenethyl isothiocyanate was activated by ascorbic acid to a greater extent than for the other two glucosinolates studied. Isothiocyanate was still the major product even at low pH although the thioglucosidase was only weakly active. Nitrile formation was always erratic in the presence of ascorbic acid. In the absence of ascorbic acid thioglucosidase was still active although to a much lesser extent, but in these circumstances benzyl thiocyanate was an additional product. There is thus a thiocyanate-forming factor in the extract of L. sativum seeds which is inactivated in the presence of ascorbic acid. This factor did not cause the formation of thiocyanate from 2-phenethylglucosinolate.     

Genetic and molecular approaches to improve nutritional value of Brassica napus L. seed

Oilseed rape (Brassica napus L.) is a major oil crop that also supplies proteins for the feed industry. In order to reduce total cost production, the objective is to increase oil yield while reducing crop inputs (especially nitrogen and pesticides). Concomitantly, it is necessary to anticipate specific uses (e.g., fatty acid composition) and to ensure the valorisation of the by-products (rapeseed meal). By the past, improvement of seed quality focused on fatty acid balance and low seed glucosinolate content. Current goals include the breeding of yellow-seeded rapeseed lines with high content of seed oil. The use of molecular tools and the exploitation of Arabidopsis knowledge will be presented and discussed.     

Modelling of the glucosinolate content in solid-state fermentation of rapeseed meal with fuzzy logic

A fuzzy model was developed to predict the glucosinolate content of rapeseed meal during solid-state fermentation. Process variables such as temperature, relative humidity, fermentation time and the resulting glucosinolate content of the meal were considered. The glucosinolate content calculated by the fuzzy model corresponded with the experimental results obtained by solid-state fermentation.     

芥菜型和甘蓝型油菜及其杂种后代种子硫甙组成差异的研究

用高效液相色谱技术分析了芥菜型油菜（Ｂｒａｓｓｉｃａ ｊｕｎｃｅａ）和甘蓝型油菜（Ｂｒａｓｓｉｃａ ｎａｐｕｓ）种间杂种后代及共亲本的硫甙组成。结果表明,与亲本农艺性状相似的后代,那么其硫甙组成与亲本亦相似。各后代中硫甙组成与亲本相比,虽发生了不同管理的变化,但都有倾向母体的趋势。芥菜型油菜烯丙基硫甙含量,２－羟基－３－丁烯基硫甙含量低,而甘蓝型油菜２－羟基３－丁烯基硫甙含量高,烯丙基硫甙含量最低     

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.     

The cabbage aphid: a walking mustard oil bomb

The cabbage aphid, Brevicoryne brassicae, has developed a chemical defence system that exploits and mimics that of its host plants, involving sequestration of the major plant secondary metabolites (glucosinolates). Like its host plants, the aphid produces a myrosinase (beta-thioglucoside glucohydrolase) to catalyse the hydrolysis of glucosinolates, yielding biologically active products. Here, we demonstrate that aphid myrosinase expression in head/thoracic muscle starts during embryonic development and protein levels continue to accumulate after the nymphs are born. However, aphids are entirely dependent on the host plant for the glucosinolate substrate, which they store in the haemolymph. Uptake of a glucosinolate (sinigrin) was investigated when aphids fed on plants or an in vitro system and followed a different developmental pattern in winged and wingless aphid morphs. In nymphs of the wingless aphid morph, glucosinolate level continued to increase throughout the development to the adult stage, but the quantity in nymphs of the winged form peaked before eclosion (at day 7) and subsequently declined. Winged aphids excreted significantly higher amounts of glucosinolate in the honeydew when compared with wingless aphids, suggesting regulated transport across the gut. The higher level of sinigrin in wingless aphids had a significant negative impact on survival of a ladybird predator. Larvae of Adalia bipunctata were unable to survive when fed adult wingless aphids from a 1% sinigrin diet, but survived successfully when fed aphids from a glucosinolate-free diet (wingless or winged), or winged aphids from 1% sinigrin. The apparent lack of an effective chemical defence system in adult winged aphids possibly reflects their energetic investment in flight as an alternative predator avoidance mechanism.     

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

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

Thioglucosidase activity from Sphingobacterium sp. strain OTG1

Screening for novel thioglucoside hydrolase activity resulted in the isolation of Sphingobacterium sp. strain OTG1 from enrichment cultures containing octylthioglucoside (OTG). OTG was hydrolysed into octanethiol and glucose by cell free extracts. Besides thioglucoside hydrolysis, several other glucoside hydrolase activities were detected in the Sphingobacterium sp. strain OTG1 cell free extract. By adding beta-glucosidase inhibitors it was possible to discriminate between these different activities. Ascorbic acid and D-gluconic acid lactone inhibited the hydrolysis of p-nitrophenyl beta-glucoside, but did not affect octyl- and octylthioglucoside hydrolase activity. Besides OTG, various other thioglucosides were hydrolysed by the novel thioglucosidase, with almost the same activities regardless of the nature of the aglycone, including the myrosinase model substrate sinigrin (a glucosinolate). Sinigrin could also be used as a growth substrate by Sphingobacterium sp. strain OTG1, although at concentrations exceeding 0.15 mM degradation was not complete.     

Effects of pH and ascorbate on benzylglucosinolate degradation in seed extracts of Lepidium sativum

The effects of pH on the enzymic degradation of benzylglucosinolate in Lepidium sativum seed autolysates were investigated both with and without addition of the enzyme co-factor ascorbic acid. Benzyl cyanide, isothiocyanate, thiocyanate and alcohol were identified in autolysates, although only traces of the alcohol were obtained. The nitrile was always the major product (80% of total glucosinolate products) even at pH 8 and 9 when the usually accepted, proton-dependent mechanism of nitrile production cannot be operative. Thiocyanate was always the second most abundant product. In the absence of added ascorbate, isothiocyanate production decreased with increasing pH, again contrary to accepted theory. L. sativum seeds thus constitute an inherently nitrile-producing system which exhibits ‘anomalous’ glucosinolate degradation. In the absence of added ascorbate, thiocyanate was the only product which was formed in approximately constant amounts, whatever the pH, so its mechanism of production is not necessarily pH-dependent. The presence of added ascorbate in general promoted enzyme activity and showed a maximum effect at ca pH 5, although minimum isothiocyanate formation was observed at that pH. At pH 4 and below, there was less glucosinolate degradation in the presence of added ascorbate than in its absence, and the conclusion is reached that at relatively high acidities the enzyme co-factor behaves as an inhibitor.     

Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low-seed,high-leaf glucosinolate oilseed mustard with uncompromised defence and yield

Glucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein-rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9-based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR-edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR-edited lines displayed a defence response at par or better than that of the wild-type line. The GTR-edited lines were equivalent to the wild-type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low-seed, high-leaf glucosinolate lines with an uncompromised defence and yield.     

Direct Bio-Utilization of Untreated Rapeseed Meal for Effective Iturin A Production by Bacillus subtilis in Submerged Fermentation

The feasibility of using untreated rapeseed meal as a nitrogen source for iturin A production by Bacillus subtilis 3–10 in submerged fermentation was first evaluated by comparison with two different commercial nitrogen sources of peptone and ammonium nitrate. A significant promoting effect of rapeseed meal on iturin A production was observed and the maximum iturin A concentration of 0.60 g/L was reached at 70 h, which was 20% and 8.0 fold higher than that produced from peptone and ammonium nitrate media, respectively. It was shown that rapeseed meal had a positive induction effect on protease secretion, contributing to the release of soluble protein from low water solubility solid rapeseed meal for an effective supply of available nitrogen during fermentation. Moreover, compared to raw rapeseed meal, the remaining residue following fermentation could be used as a more suitable supplementary protein source for animal feed because of the great decrease of major anti-nutritional components including sinapine, glucosinolate and its degradation products of isothiocyanate and oxazolidine thione. The results obtained from this study demonstrate the potential of direct utilization of low cost rapeseed meal as a nitrogen source for commercial production of iturin A and other secondary metabolites by Bacillus subtilis.     

Studies on glucosinolate degradation in Lepidium sativum seed extracts

Analysis of Lepidium sativum seeds showed the presence of allyl, 2-phenethyl and benzyl glucosinolates, the first two being reported for the first time from this source. The effects of temperature, pH of the extraction medium and the length of time allowed for autolysis were assessed on the benzyl glucosinolate degradation products in seed extracts. In particulàr benzyl thiocyanate was not produced at higher temperatures but at ambient and lower temperatures it exceeded isothiocyanate. Nitrile was always the major product under the conditions studied, ever at pH levels as high as 7.4. Five new possible benzyl glucosinolate degradation products were detected and evidence is presented that benzaldehyde and benzyl alcohol could be secondary products formed thermally from isothocyanate and thiocyanate, respectively. Benzyl mercaptan and benzyl methyl sulphide also appear to be thermally produced.