Synthesis and anti-inflammatory activity of aromatic glucosinolates

Aromatic GLs are important members of the glucosinolate family of compounds because of their potential biological activity and medicinal properties. This study has shown success in the high yielding synthesis of some important aromatic GLs as well as the results of testing for anti-inflammatory properties of the synthetic GLs. 3,4-Dimethoxyphenylglucosinolate was found to be the most active anti-inflammatory of the seven glucosinolates assayed.     

Integration of Biosynthesis and Long-Distance Transport Establish Organ-Specific Glucosinolate Profiles in Vegetative Arabidopsis

Although it is essential for plant survival to synthesize and transport defense compounds, little is known about the coordination of these processes. Here, we investigate the above- and belowground source-sink relationship of the defense compounds glucosinolates in vegetative Arabidopsis thaliana. In vivo feeding experiments demonstrate that the glucosinolate transporters1 and 2 (GTR1 and GTR2), which are essential for accumulation of glucosinolates in seeds, are likely to also be involved in bidirectional distribution of glucosinolates between the roots and rosettes, indicating phloem and xylem as their transport pathways. Grafting of wild-type, biosynthetic, and transport mutants show that both the rosette and roots are able to synthesize aliphatic and indole glucosinolates. While rosettes constitute the major source and storage site for short-chained aliphatic glucosinolates, long-chained aliphatic glucosinolates are synthesized both in roots and rosettes with roots as the major storage site. Our grafting experiments thus indicate that in vegetative Arabidopsis, GTR1 and GTR2 are involved in bidirectional long-distance transport of aliphatic but not indole glucosinolates. Our data further suggest that the distinct rosette and root glucosinolate profiles in Arabidopsis are shaped by long-distance transport and spatially separated biosynthesis, suggesting that integration of these processes is critical for plant fitness in complex natural environments.     

Naturally occurring glucosinolates and isothiocyanates as a weapon against chronic pain: potentials and limits

Phytochemistry Reviews - Investigation into glucosinolates (GLs) therapeutic effects boasts a long history, which began with the evidence that their hydrolysis-derived isothiocyanates (ITCs) could...     

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.     

Indole glucosinolate breakdown and its biological effects

Most species in the Brassicaceae produce one or more indole glucosinolates. In addition to the parent indol-3-ylmethylglucosinolate (IMG), other commonly encountered indole glucosinolates are 1-methoxyIMG, 4-hydroxyIMG, and 4-methoxyIMG. Upon tissue disruption, enzymatic hydrolysis of IMG produces an unstable aglucone, which reacts rapidly to form indole-3-acetonitrile and indol-3-ylmethyl isothiocyanate. The isothiocyanate, in turn, can react with water, ascorbate, glutathione, amino acids, and other plant metabolites to produce a variety of physiologically active indole compounds. Myrosinase-initiated breakdown of the substituted indole glucosinolates proceeds in a similar manner to that of IMG. Induction of indole glucosinolate production in response to biotic stress, experiments with mutant plants, and artificial diet assays suggest a significant role for indole glucosinolates in plant defense. However, some crucifer-feeding specialist herbivores recognize indole glucosinolates and their breakdown products as oviposition and/or feeding stimulants. In mammalian diets, IMG can have both beneficial and deleterious effects. Most IMG breakdown products induce the synthesis of phase 1 detoxifying enzymes, which may in some cases prevent carcinogenesis, but in other cases promote carcinogenesis. Recent advances in indole glucosinolate research have been fueled by their occurrence in the well-studied model plant Arabidopsis thaliana. Knowledge gained from genetic and biochemical experiments with A. thaliana can be applied to gain new insight into the ecological and nutritional properties of indole glucosinolates in other plant species.     

Cytochromes P450 in the biosynthesis of glucosinolates and indole alkaloids

Characteristic of cruciferous plants is the synthesis of nitrogen- and sulfur-rich compounds, such as glucosinolates and indole alkaloids. The intact glucosinolates have limited biological activity, but give rise to an array of bio-active breakdown products when hydrolysed by endogenous β-thioglucosidases (myrosinases) upon tissue disruption. Both glucosinolates and indole alkaloids constitute an important part of the defence of plants against herbivores and pathogens, with the difference that a basal level of glucosinolates is ever-present in the plant whereas indole alkaloids are true phytoalexins that are de novo synthesised upon pathogen attack. With the completion of the genome sequence of the model plant, Arabidopsis thaliana, which is a crucifer, many genes involved in the biosynthesis of glucosinolates and indole alkaloids have been identified and cytochromes P450 are key players in these pathways. In the present review, we will focus on the cytochromes P450 in the biosynthesis of both groups of compounds. Their functional roles and regulation will be discussed.     

Identification of indole glucosinolate breakdown products with antifeedant effects on Myzus persicae (green peach aphid)

The cleavage of glucosinolates by myrosinase to produce toxic breakdown products is a characteristic insect defense of cruciferous plants. Although green peach aphids ( Myzus persicae ) are able to avoid most contact with myrosinase when feeding from the phloem of Arabidopsis thaliana , indole glucosinolates are nevertheless degraded during passage through the insects. A defensive role for indole glucosinolates is suggested by the observation that atr1D mutant plants, which overproduce indole glucosinolates, are more resistant to M. persicae , whereas cyp79B2 cyp79B3 double mutants, which lack indole glucosinolates, succumb to M. persicae more rapidly. Indole glucosinolate breakdown products, including conjugates formed with ascorbate, glutathione and amino acids, are elevated in the honeydew of M. persicae feeding from atr1D mutant plants, but are absent when the aphids are feeding on cyp79B2 cyp79B3 double mutants. M. persicae feeding from wild-type plants and myrosinase-deficient tgg1 tgg2 double mutants excrete a similar profile of indole glucosinolate-derived metabolites, indicating that the breakdown is independent of these foliar myrosinases. Artificial diet experiments show that the reaction of indole-3-carbinol, a breakdown product of indol-3-ylmethylglucosinolate, with ascorbate, glutathione and cysteine produces diindolylmethylcysteines and other conjugates that have antifeedant effects on M. persicae . Therefore, the post-ingestive breakdown of indole glucosinolates provides a defense against herbivores such as aphids that can avoid glucosinolate activation by plant myrosinases.     

植物激素与芥子油苷在生物合成上的相互作用

植物激素在植物的生长发育中起着关键性作用,芥子油苷是一类重要的次生代谢物质。植物激素与芥子油苷之间存在复杂的相互作用。生长素与吲哚类芥子油苷在生物合成上存在着相互作用。植物防卫信号分子与芥子油苷之间也存在相互作用,茉莉酸强烈诱导吲哚类芥子油苷生物合成相关基因CYP7982和CYP7983的表达,从而诱导吲哚-3-甲基芥子油苷和N-甲氧吲哚-3-甲基芥子油苷等吲哚类芥子油苷的生成,水杨酸和乙烯则能轻度诱导4-甲氧吲哚-3-甲基芥子油苷的生成。植物防卫信号转导途径相互作用以精细调节不同种类吲哚类芥子油苷的生成。     

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.     

Indole Glucosinolates and Camalexin do not Influence the Development of the Clubroot Disease in Arabidopsis thaliana

Root galls of Brassicaceae caused by Plasmodiophora brassicae are dependent on increased auxin and cytokinin formation. In this study we investigated whether indole glucosinolates are involved in indole‐3‐acetic acid (IAA) biosynthesis in root galls, by using a genetic approach. The cytochrome P450 enzymes, CYP79B2 and CYP79B3, convert tryptophan to indole‐3‐acetaldoxime (IAOx), which is a precursor for indole glucosinolates and the phytoalexin camalexin in Arabidopsis thaliana. Root galls of the Arabidopsis ecotypes Wassilewskija (WS) and Columbia (Col) accumulated camalexin, WS at levels up to 320 μg/g dry weight. By contrast, camalexin was absent in root galls of cyp79b2/b3 double mutants. Infection rate and disease index as a measure of club development in mutant and wild‐type plants of the two ecotypes were investigated and no differences were found in gall formation. This demonstrates that camalexin is an ineffective inhibitor of P. brassicae and indole glucosinolates are not the source of elevated levels of IAA in galls, because free IAA levels in mutant galls were comparable with those in wild type.     

Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification

Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.     

Myzus persicae (green peach aphid) feeding on Arabidopsis induces the formation of a deterrent indole glucosinolate

Cruciferous plants produce a wide variety of glucosinolates as a protection against herbivores and pathogens. However, very little is known about the importance of individual glucosinolates in plant defense and the regulation of their production in response to herbivory. When Myzus persicae (green peach aphid) feeds on Arabidopsis aliphatic glucosinolates pass through the aphid gut intact, but indole glucosinolates are mostly degraded. Although aphid feeding causes an overall decrease in Arabidopsis glucosinolate content, the production of 4-methoxyindol-3-ylmethylglucosinolate is induced. This altered glucosinolate profile is not a systemic plant response, but is limited to the area in which aphids are feeding. Aphid feeding on detached leaves causes a similar change in the glucosinolate profile, demonstrating that glucosinolate transport is not required for the observed changes. Salicylate-mediated signaling has been implicated in other plant responses to aphid feeding. However, analysis of eds5, pad4, npr1 and NahG transgenic Arabidopsis, which are compromised in this pathway, demonstrated that aphid-induced changes in the indole glucosinolate profile were unaffected. The addition of purified indol-3-ylmethylglucosinolate to the petioles of cyp79B2 cyp79B3 mutant leaves, which do not produce indole glucosinolates, showed that this glucosinolate serves as a precursor for the aphid-induced synthesis of 4-methoxyindol-3-ylmethylglucosinolate. In artificial diets, 4-methoxyindol-3-ylmethylglucosinolate is a significantly greater aphid deterrent in the absence of myrosinase than its metabolic precursor indol-3-ylmethylglucosinolate. Together, these results demonstrate that, in response to aphid feeding, Arabidopsis plants convert one indole glucosinolate to another that provides a greater defensive benefit.     

Modulation of CYP79 genes and glucosinolate profiles in Arabidopsis by defense signaling pathways

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.     

盐胁迫对拟南芥和盐芥莲座叶芥子油苷含量的影响

芥子油苷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。以模式植物拟南芥（Arabidopsis thaliana）和盐生模式植物盐芥（Thellungiella halophila）为研究对象,系统地分析了盐胁迫下二者芥子油苷组成和含量的变化规律。拟南芥（生长4周）和盐芥（生长6周）叶片的芥子油苷组成在盐胁迫后没有改变。拟南芥的芥子油苷总量、脂肪族芥子油苷总量、吲哚族芥子油苷总量受盐胁迫的影响均不显著,而盐芥的则随盐胁迫增强先减少、后增加并高于对照水平。拟南芥脂肪族的3MSOP、5MSOP和吲哚族的4OHI3M、4MOI3M随盐胁迫增强而含量降低,而脂肪族的6MSOH、吲哚族的I3M以及盐芥脂肪族的3MSOP则随盐胁迫增强有含量增加的趋势。拟南芥脂肪族的8MSOO和吲哚族的1MOI3M,盐芥脂肪族的3MTP、Allyl、10MSD和吲哚族的4MOI3M,在盐胁迫下的含量变化与盐芥芥子油苷总量的变化趋势一致。     

油菜硫苷组分含量及抑菌活性研究

对油菜不同器官硫苷的组分与含量进行分析,结果表明根系和茎中的硫苷总量显著高于叶片,根系中的硫苷以4甲-氧基-3吲-哚甲基硫苷和1-甲氧基-3吲-哚甲基硫苷为主,茎和叶中以2羟-基-3丁-烯基硫苷和苯乙基硫苷为主。油菜根系中的硫苷组分从冬前期到终花期基本保持稳定,硫苷各组分的含量在不同生育时期的变化基本一致,终花期含量最高,成熟期含量最低。19个油菜品种根系中硫苷组分与含量存在较大差异;以草莓灰霉菌为靶标物,研究油菜根系的挥发性水解产物和水溶性水解产物对土传病原真菌的抑制作用,发现不同油菜品种根系水解产物的抑菌活性与其硫苷组分与含量有关,草莓灰霉菌对丁烯基ITC非常敏感,吲哚类ITCs对草莓灰霉生长的抑制作用要低于芳香族ITCs。     

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.     

CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis

In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K(m) value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.     

Localization of the glucosinolate biosynthetic enzymes reveals distinct spatial patterns for the biosynthesis of indole and aliphatic glucosinolates

Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore‐tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense‐related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.     

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。