Seasonal variation in glucosinolate content in Brassica oleracea crops grown in northwestern Spain

Brassica oleracea L. crops including kales, cabbages, and Tronchuda cabbages are widely grown in northwestern Spain and Portugal but little information is available on leaf glucosinolate content of these crops. The objectives were to determine the diversity for the total glucosinolate content and profile on leaves in a collection of 153 kales, 26 cabbages, and three Tronchuda cabbages varieties grown at two growing seasons and to determine the seasonal variation of glucosinolates in cabbages and Tronchuda cabbage varieties. Sinigrin, glucoiberin, and glucobrassicin were the major glucosinolates found in kales. Glucoiberin was the most common glucosinolate in Tronchuda cabbages in both planting seasons and in cabbages sown in fall season whereas glucobrassicin and glucoiberin were the most common glucosinolates in cabbages in spring season. In kales the total glucosinolate content ranged from 11.0 to 53 micromol g(-1) dw, with a mean value of 26.3 micromol g(-1) dw. Four kale varieties (MBG-BRS0468, MBG-BRS0476, MBG-BRS0060 and MBG-BRS0223) showed the highest total sinigrin or glucobrassicin contents. So, they could be good candidates for future breeding programs. In cabbages, the total glucosinolate content ranged from 10.9 to 27 g(-1) dw. Total glucosinolate concentration during spring sowing (22 micro mg(-1) dw) was higher than those in fall sowing (13 micro mg(-1) dw). Regarding both high glucosinolate content and the agronomic value, MBG-BRS0057 and MBG-BRS0074 could be good sources of beneficial glucosinolates. The presence of high concentrations of sinigrin, glucoiberin, and glucobrassicin warrant further search into their potential use to enhance the level of these important phytochemicals in these edible crops.     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.     

Quantification of glucosinolates in leaves of leaf rape (Brassica napus ssp. pabularia) by near-infrared spectroscopy

The potential of near-infrared spectroscopy (NIRS) for screening the total glucosinolate (t-GSL) content, and also, the aliphatic glucosinolates gluconapin (GNA), glucobrassicanapin (GBN), progoitrin (PRO), glucoalyssin (GAL), and the indole glucosinolate glucobrassicin (GBS) in the leaf rape (Brassica napus L. ssp. pabularia DC), was assessed. This crop is grown for edible leaves for both fodder and human consumption. In Galicia (northwestern Spain) it is highly appreciated for human nutrition and have the common name of "nabicol". A collection of 36 local populations of nabicol was analysed by NIRS for glucosinolate composition. The reference values for glucosinolates, as they were obtained by high performance liquid chromatography on the leaf samples, were regressed against different spectral transformations by modified partial least-squares (MPLS) regression. The coefficients of determination in cross-validation (r2) shown by the equations for t-GSL, GNA, GBN, PRO, GAL and GBS were, respectively, 0.88, 0.73, 0.81, 0.78, 0.37 and 0.41. The standard deviation to standard error of cross-validation ratio, were for these constituents, as follows: t-GSL, 2.96; GNA, 1.94; GBN, 2.31; PRO, 2.11; GAL, 1.27, and GBS, 1.29. These results show that the equations developed for total glucosinolates, as well as those for gluconapin, glucobrassicanapin and progoitrin, can be used for screening these compounds in the leaves of this species. In addition, the glucoalyssin and glucobrassicin equations obtained, can be used to identify those samples with low and high contents. From the study of the MPLS loadings of the first three terms of the different equations, it can be concluded that some major cell components as protein and cellulose, highly participated in modelling the equations for glucosinolates.     

Glucosinolate changes in developing pods of single and double low varieties of autumn-sown oilseed rape (B. napus)

Single and double low varieties of oilseed rape were grown in the 1987/88 and 1988/89 seasons to study changes in the concentrations of total and individual glucosinolates within pods during development. Total glucosinolate concentration in seeds of all varieties increased during development when expressed on a fresh weight basis. The levels of the major alkenyl glucosinolates present in the seed; 2–hydroxy-3–butenyl, 3–butenyl and 4–pentenyl had been reduced in the transition from single to double low varieties. The major indole glucosinolates in the seed, 4–hydroxy-3–indolylmethyl and 3–indolylmethyl were present in the same amounts in single and double low varieties but in the latter represented a greater proportion of the total seed glucosinolate content. A decline in the total glucosinolate concentration in the pod walls with time together with the analogous profile of individual glucosinolates in the seeds and pod walls suggests that the pod wall is a major site of seed glucosinolate synthesis. Other plant parts may also have an important role to play in provision of intact glucosinolates or precursors to the pod walls for glucosinolate biosynthesis.     

Variation of glucosinolates on position orders of flower buds in turnip rape (Brassica rapa)

To glucosinolate (GSL) contents on flower buds depending on their position orders in turnip rape (Brassica rapa), three Japanese ‘Nabana’ cultivars such as cv. No. 21 (Brassica rapa, early type), cv. Husanohana (B. rapa, late type) and cv. Norin No. 20 (B. napus) were investigated using HPLC analysis. Ten GSLs including glucoraphanin, sinigrin, glucoalyssin, napoleiferin, gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin, and gluconasturtiin were detected. Differences in individual and total GSL contents were found between two plant varieties, which are also depending on various developmental stages. Among the GSLs, gluconapin (mean 23.11 μmole/g dry weight (DW) and glucobrassicanapin (mean 13.41 μmole/g DW) documented the most abundant compounds and contributed average 39 and 27% of the total GSLs, but indolyl and aromatic GSLs together accounted >10% of the total GSLs. The presence of significant quantities of gluconapin in the cultivars should be studied more extensively, since the GSL is mainly responsible for the bitter taste.     

Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana

The glucosinolate content of various organs of the model plant Arabidopsis thaliana (L.) Heynh., Columbia (Col-0) ecotype, was analyzed at different stages during its life cycle. Significant differences were noted among organs in both glucosinolate concentration and composition. Dormant and germinating seeds had the highest concentration (2.5-3.3% by dry weight), followed by inflorescences, siliques (fruits), leaves and roots. While aliphatic glucosinolates predominated in most organs, indole glucosinolates made up nearly half of the total composition in roots and late-stage rosette leaves. Seeds had a very distinctive glucosinolate composition. They possessed much higher concentrations of several types of aliphatic glucosinolates than other organs, including methylthioalkyl and, hydroxyalkyl glucosinolates and compounds with benzoate esters than other organs. From a developmental perspective, older leaves had lower glucosinolate concentrations than younger leaves, but this was not due to decreasing concentrations in individual leaves with age (glucosinolate concentration was stable during leaf expansion). Rather, leaves initiated earlier in development simply had much lower rates of glucosinolate accumulation per dry weight gain throughout their lifetimes. During seed germination and leaf senescence, there were significant declines in glucosinolate concentration. The physiological and ecological significance of these findings is briefly discussed.     

拟南芥莲座叶芥子油苷含量对水分胁迫的响应

芥子油苷(glucosinolates)是十字花科植物中一类含氮、含硫的次生代谢产物,与其水解产物在植物防御功能中有重要意义且与环境因子关系密切.通过控制供水的方式对营养生长时期的拟南芥幼苗进行水分胁迫,观察了土壤自然干旱对营养生长时期拟南芥莲座叶芥子油苷含量及组成的影响.结果表明,土壤自然干旱处理下,拟南芥莲座叶的芥子油苷总量从处理3 d起低于对照,且随着处理天数的增加与对照组的差异逐渐增大,脂肪族芥子油苷的响应均比较明显,与芥子油苷总量的变化趋势基本一致,而吲哚族芥子油苷对水分胁迫则不敏感.脂肪族中的4-甲基亚磺酰丁基芥子油苷(4-methylsulphinylbutyl GS,4MSOB)占脂肪族芥子油苷的比例最大,它的含量变化成为影响莲座叶中芥子油苷组合模式的主导因素.     

Oviposition and tarsal chemoreceptors of the cabbage root fly are stimulated by glucosinolates and host plant extracts

The role of glucosinolates in the oviposition behaviour of the cabbage root fly,Delia radicum (L.) (Diptera, Anthomyiidae) was investigated using egg counts and electrophysiological recordings from tarsal contact chemoreceptors. The glucosinolates present both inside and on the surface of cauliflower leaves were determined. The total amounts obtained with the two methods differed by a factor of 100. The extract of the leaf surface contained about 60 μg per g leaf extracted (gle), the total leaf extract 7.5 mg per gle. The glucosinolate patterns of the two extracts were qualitatively similar, but the ratios of the content of individual glucosinolates showed considerable differences. The D sensilla on segment 3 and 4 of the tarsus ofD. radicum females were shown to contain a sensitive receptor cell for glucosinolates. In contrast, the receptor cells of the D sensilla of the other segments did not respond in a dose dependent way to these compounds. The glucosinolate receptors were found to be especially sensitive to glucobrassicin, gluconasturtiin and glucobrassicanapin with thresholds of about 10⁻⁸ M to 10⁻⁹ M. Large differences (up to two orders of magnitude) were observed among the different glucosinolates. A significant correlation was found between the behavioural discrimination index and the electrophysiological results. But no obvious correlation existed between the chemical nature of the glucosinolate side chain (e.g. indole, aromatic and aliphatic groups), and their stimulatory activity. However, a significant correlation was found between the overall length of the side chain and the biological activity. Although the flies discriminated clearly between model leaves with and without glucosinolates, a clear dose response curve was only obtained for the indole glucosinolate glucobrassicin. Since the most stimulatory fraction of the surface extract contained no glucosinolates, it was concluded that other compounds, in addition to glucosinolates, do play an important role for the stimulation of oviposition.     

Glucosinolate responses of oilseed rape, mustard and kale to mechanical wounding and infestation by cabbage stem flea beetle (Psylliodes chrysocephala)

Mechanical wounding of the petioles of six laboratory-grown rapeseed ( Brassica napus ) cultivars induced physiological changes in the plant, markedly affecting the levels of individual glucosinolates. Greatest increases were observed for the indole glucosinolates, glucobrassicin and neoglucobrassicin. Such changes were usually associated with large decreases in the levels of aliphatic glucosinolates. The total glucosinolate content of the wounded plant was thus a reflection of these two opposing trends and wounding produced a greater relative indole glucosinolate content in this total figure. Thus increasing wounding was associated with an increase in indole glucosinolates and a decrease in aliphatic compounds.
Infestation of field- and laboratory-grown rapeseed with cabbage stem flea beetle ( Psylliodes chrysocephala ) produced similar effects, which were observed in various parts of the plant. Differences in response between field- and laboratory-grown infested plants are attributed to the different physiological ages of the harvested material.
Laboratory-grown kale and mustards also showed wound-induced glucosinolate changes. The kale, cv. Fribor, produced elevated levels of both indoles and aliphatics after wounding. Total glucosinolate content in the mustards, which, unlike rape and kale, normally contain only traces of indole glucosinolates in the unstressed state, was increased following wounding. This was, however, not associated with elevated levels of indole glucosinolates, but with accumulation of aliphatic ( Brassica nigra, B. juncea ) and aromatic ( Sinapis alba ) glucosinolates. The significance of these findings is discussed.     

Glucosinolate contents in maca (Lepidium peruvianum Chacón) seeds, sprouts, mature plants and several derived commercial products

Several products derived from processed maca hypocotyls (Lepidium peruvianum Chacón, previously known asL. meyenii Walp.) were surveyed for glucosinolate content and quantified by HPLC analysis. These included pills, capsules, flour, liquor, tonic and mayonnaise. Different plant organs such as fresh hypocotyls and leaves, seeds, dry hypocotyls, and sprouts were also included in the survey. The most abundant glucosinolates detected in fresh and dry hypocotyls and leaves were the aromatic glucosinolates, benzylglucosinolate (glucotropaeolin) and p-methoxybenzylglucosinolate. Maca seeds and sprouts differed in profile from hypocotyls and leaves due to the modification of benzylglucosinolate. No glucosinolates were detected in liquor and tonic, while mayonnaise had only trace amounts of those glucosinolates. It had instead allylglucosinolate (sinigrin), which is an aliphatic glucosinolate. The pills, capsules and flour had the same glucosinolates as those observed in hypocotyls, but in variable amounts. The richest sources of glucosinolates were seeds, fresh hypocotyls and sprouts, in that order.     

Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. Despite this importance, little is known about the regulation of secondary metabolite accumulation. We are studying the regulation of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate how secondary metabolism is controlled. We utilized Ler and Cvi, two ecotypes of Arabidopsis that have striking differences in both the types and amounts of glucosinolates that accumulate in the seeds and leaves. QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate levels. Our results show that two of the loci controlling total aliphatic glucosinolates map to biosynthetic loci that interact epistatically to regulate aliphatic glucosinolate accumulation. In addition to the six loci regulating total indolic glucosinolate concentration, mapping of QTL for the individual indolic glucosinolates identified five additional loci that were specific to subsets of the indolic glucosinolates. These data show that there are a large number of variable loci controlling glucosinolate accumulation in Arabidopsis thaliana.     

Genetic control of natural variation in Arabidopsis glucosinolate accumulation

Glucosinolates are biologically active secondary metabolites of the Brassicaceae and related plant families that influence plant/insect interactions. Specific glucosinolates can act as feeding deterrents or stimulants, depending upon the insect species. Hence, natural selection might favor the presence of diverse glucosinolate profiles within a given species. We determined quantitative and qualitative variation in glucosinolates in the leaves and seeds of 39 Arabidopsis ecotypes. We identified 34 different glucosinolates, of which the majority are chain-elongated compounds derived from methionine. Polymorphism at only five loci was sufficient to generate 14 qualitatitvely different leaf glucosinolate profiles. Thus, there appears to be a modular genetic system regulating glucosinolate profiles in Arabidopsis. This system allows the rapid generation of new glucosinolate combinations in response to changing herbivory or other selective pressures. In addition to the qualitative variation in glucosinolate profiles, we found a nearly 20-fold difference in the quantity of total aliphatic glucosinolates and were able to identify a single locus that controls nearly three-quarters of this variation.     

外源喷施萘乙酸对小白菜硫代葡萄糖苷的影响

本试验研究了不同浓度的萘乙（NAA）并结合单次喷施（NAA-1）和两次喷施（NAA．2）,对小白菜生长和硫代葡萄糖苷（简称硫苷）含量的影响。结果表明,与对照相比,不同的NAA处理浓度均显著增加了小白菜的鲜重。同时,NAA处理对总硫苷和单个硫苷含量产生了显著的影响。NAA-1处理时,总脂肪族硫苷、2．苯乙基硫苷和总硫苷在20mg·L-1时到达最大值;而总吲哚族硫苷在50mg·L-1时含量到达最高。NAA．2处理时,大部分单个硫苷和总硫苷在10mg·L-1处理即达到最大值。可见,在较低浓度两次喷施NAA试验中对大部分硫苷的诱导效果高于单次喷施NAA;但随着NAA处理浓度的提高,单次喷施NAA对硫苷的诱导效果较好。其中,吲哚-3-甲基硫苷含量在NAA-1处理,喷施50mg·L-1时显著高于其他处理：而2-苯乙基硫苷在单次和两次喷施NAA时,分别在20mg·L-1和10mg·L-达到了最大值。     

Resistance of cabbage (Brassica oleracea capitata group) crops to Mamestra brassicae

Twenty-one cabbage (Brassica oleracea capitata group) varieties, including 16 local varieties and five commercial hybrids, were screened for resistance to the moth Mamestra brassicae L. under natural and artificial conditions in northwestern Spain. Resistance was assessed as the proportion of damaged plants and damaged leaves, leaf feeding injury, and number of larvae present. Correlation coefficients among damage traits showed that a visual scale (general appearance rating) should be a useful indicator of resistance. Most local varieties were highly susceptible to M. brassicae, whereas the commercial hybrids tested were resistant in terms of head foliage consumption and number of larvae per plant. Performance of varieties was similar under natural and artificial infestation although some of them performed differently at each year. Three local varieties (MBG-BRS0057, MBG-BRS0074, and MBG-BRS0452) were highly susceptible at both natural and artificial infestation conditions being MBG-BRS0074 the most damaged variety. Two local varieties (MBG-BRS0402 and MBG-BRS0535) and commercial hybrids were identified as resistant or moderately resistant to M. brassicae. Among them, 'Corazón de Buey' and 'Cabeza negra' were the most resistant and produced compact heads. These varieties could be useful sources of resistance to obtain resistant varieties to M. brassicae or as donors of resistance to other Brassica crops. The possible role of leaf traits, head compactness, and leaf glucosinolate content in relation to M. brassicae resistance is discussed.     

Quantitative trait loci analysis of non-enzymatic glucosinolate degradation rates in Brassica oleracea during food processing

Epidemiological and mechanistic studies show health-promoting effects of glucosinolates and their breakdown products. In literature, differences in non-enzymatic glucosinolate degradation rates during food processing between different vegetables are described, which provide the basis for studying the genetic effects of this trait and breeding vegetables with high glucosinolate retention during food processing. Non-enzymatic glucosinolate degradation, induced by heat, was studied in a publicly available Brassica oleracea doubled haploid population. Data were modeled to obtain degradation rate constants that were used as phenotypic traits to perform quantitative trait loci (QTL) mapping. Glucosinolate degradation rate constants were determined for five aliphatic and two indolic glucosinolates. Degradation rates were independent of the initial glucosinolate concentration. Two QTL were identified for the degradation rate of the indolic glucobrassicin and one QTL for the degradation of the aliphatic glucoraphanin, which co-localized with one of the QTL for glucobrassicin. Factors within the plant matrix might influence the degradation of different glucosinolates in different genotypes. In addition to genotypic effects, we demonstrated that growing conditions influenced glucosinolate degradation as well. The study identified QTL for glucosinolate degradation, giving the opportunity to breed vegetables with a high retention of glucosinolates during food processing, although the underlying mechanisms remain unknown.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

Suppressive Effect of Yamato-mana (Brassica rapa L. Oleifera Group) Constituent 3-Butenyl Glucosinolate (Gluconapin) on Postprandial Hypertriglyceridemia in Mice

We examined the bioactivity of Yamato-mana (Brassica rapa L. Oleifera Group) constituent glucosinolates and found that 3-butenyl glucosinolate (gluconapin) decreased the plasma triglyceride gain induced by corn oil administration to mice. However, phenethyl glucosinolate (gluconasturtiin) had little effect. 2-Propenyl glucosinolate (sinigrin) also reduced the plasma triglyceride level, which suggests that alkenyl glucosinolates might be promising agents to prevent postprandial hypertriglyceridemia.