Glucosinolates in <Emphasis Type="Italic">Brassica</Emphasis> foods: bioavailability in food and significance for human health

Glucosinolates are sulphur compounds that are prevalent in Brassica genus. This includes crops cultivated as vegetables, spices and sources of oil. Since 1970s glucosinolates and their breakdown products, have been widely studied by their beneficial and prejudicial biological effects on human and animal nutrition. They have also been found to be partly responsible for the characteristic flavor of Brassica vegetables. In recent years, considerable attention has been paid to cancer prevention by means of natural products. The cancer-protective properties of Brassica intake are mediated through glucosinolates. Isothyocianate and indole products formed from glucosinolates may regulate cancer cell development by regulating target enzymes, controlling apoptosis and blocking the cell cycle. Nevertheless, variation in content of both glucosinolates and their bioactive hydrolysis products depends on both genetics and the environment, including crop management practices, harvest and storage, processing and meal preparation. Here, we review the significance of glucosinolates as source of bioactive isothiocyanates for human nutrition and health and the influence of environmental conditions and processing mechanisms on the content of glucosinolate concentration in Brassica vegetables. Currently, this area is only partially understood. Further research is needed to understand the mechanisms by which the environment and processing affect glucosinolates content of Brassica vegetables. This will allow us to know the genetic control of these variables, what will result in the development of high quality Brassica products with a health-promoting activity.     

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.     

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.     

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.     

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.     

The characterisation of <Emphasis Type="Italic">AOP2</Emphasis>: a gene associated with the biosynthesis of aliphatic alkenyl glucosinolates in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Glucosinolates, a group of nitrogen and sulfur containing compounds associated with plant-insect interactions, are produced by a number of important Brassicaceae crop species. In Arabidopsis the AOP2 gene plays a role in the secondary modification of aliphatic (methionine-derived) glucosinolates, namely the conversion of methylsulfinylalkyl glucosinolates to form alkenyl glucosinolates, and also influences aliphatic glucosinolate accumulation.     

Leaf surface wax layers of Brassicaceae lack feeding stimulants for Phaedon cochleariae

Numerous reports have indicated that glucosinolates are important stimulants for specialist herbivores feeding on Brassicaceae, and that these metabolites might be present on the plant surface and thereby detectable by an alighting insect. We investigated the outermost layer of leaves of two species of Brassicaceae, Brassica napus L. var. ‘Martina’ and Nasturtium officinale R. Br., using two highly selective extraction methods. When the epicuticular wax layer was mechanically removed with gum arabic, no glucosinolates were detectable in the lower and upper leaf surfaces. Extracting the leaf surfaces with a threefold short rinse with chloroform/methanol/water (2 : 1 : 1 vol/vol/vol) led to varying results, depending on the light conditions under which plants had been kept in the period prior to extraction. In plants kept under light, glucosinolates were detectable in a first extraction in minor concentrations, with increasing amounts in a second and third extraction. In plants kept in darkness, glucosinolates were almost absent in the first extraction. We postulate that the polar glucosinolates are washed from the inner leaf tissue through open stomata to the outside during solvent extraction, but are not naturally present in the outermost wax layer. The response of the crucifer specialist Phaedon cochleariae (F.) (Coleoptera: Chrysomelidae) to leaf surfaces of the host plants B. napus and N. officinale and to a glucosinolate was tested. Adults preferred both the adaxial and abaxial leaf surfaces of host plants that had been treated with gum arabic in order to remove the epicuticular waxes over intact surfaces. Waxes may therefore prevent direct contact with the stimulants. Sinigrin (allyl glucosinolate) and/or surface extracts of N. officinale leaves applied on Pisum sativum leaf discs did not evoke feeding, but feeding did occur when total leaf extracts of B. napus or N. officinale were applied on this non‐host. We conclude that glucosinolates might only act as feeding stimulants for P. cochleariae in concert with compounds other than surface waxes.     

Tarsal contact chemoreceptor response to glucosinolates and cardenolides mediating oviposition in Pieris rape

Abstract. Water-soluble extracts of a host crucifer (Brassica oleracea L.) and non-host crucifer (Erysimum cheiranthoides L.) and isolated pure cardenolides and glucosinolates were tested on Pieris rapae L. (Lepidoptera: Pieridae) butterflies in oviposition assays and by electrophysiological recordings from the contact-chemoreceptor sensilla of the prothoracic tarsi. Ten different glucosinolates stimulated oviposition to varying degree when put on non-host plant leaves. The most active compounds were glucobrassiein and gluconasturtiin (methylindole and phenylethyl aglycone), whereas glucocapparin, sinalbin. glucotropaeolin, sinigrin and glucoalyssinin had intermediate activity. Glucocheirolin, glucoerucin and glucoiberin (sulphur-containing aglycone) were significantly less active. Of eight cardenolides applied to host-plant leaves (100 μg each), four glycosides deterred oviposition strongly (erysimoside, erychroside, cymarin and K-strophanthin-β). Erycordin, helveticoside, digitoxin and strophanthidin had little or no deterrent activity. Sensilla located laterally on the prothoracic tarsi of female butterflies contained one receptor cell sensitive to sucrose. None of the tested extracts or pure compounds stimulated any cell in these sensilla. In contrast, the cells in the medial sensilla showed little or no sensitivity to sucrose. One cell was found to be sensitive to cardenolide glycosides. The threshold for one of the most active compounds, erychroside, was about 0.1 μg/ml (1.5 times 10^-7 M). Two receptor cells, characterized by spikes of differing amplitude, were sensitive to glucosinolates. One of these, with larger amplitude spikes, seemed to be the same as the cell sensitive to cardenolides. The threshold for the most active glucosinolates, glucobrassicin and gluconasturtiin was estimated to be below 0.1 mg/ml (2 times 10^-4 M). The neural activity of both classes of compounds, cardenolides and glucosinolates, was significantly correlated with their behavioural activity as deterrents or stimulants.     

The phloem mobility of glucosinolates

The transport properties of glucosinolates within Brassica napus are of interest as identification of the mechanism of transport could lead to lower levels being obtained in specific tissues such as the seeds. The phloem mobility of ³⁵S-gluconapin (but-3-enylglucosinolate) and ³⁵S-desulphogluconapin in oilseed rape plants has been inferred from tissue distribution patterns, as well as from observed coincident phloem mobility of ³H-gluconapin and ¹⁴C-sucrose. The measured relative phloem mobilities for sinigrin (2-propenylglucosinolate), ³H-gluconapin, ³⁵S-desulphogluconapin, ³⁵S-desulphosinigrin, ¹⁴C-tryptophan, ³H-AIB (-aminoisobutyric acid), and literature values for a reduced ³H-oligogalacutonide elicitor (degree of polymerization 6) and ¹⁴C-IAA (indolylacetic acid), have been compared with the predicted values obtained using the Kleier model for phloem mobility of xenobiotics. All the above compounds show phloem systemicity, demonstrated using the Ricinus assay, as predicted by the model. Log Kow (octanol-water partition coefficient) values for glucosinolates and desulphoglucosinolates measured at pH 4 and pH 7, and the effect of pH on uptake by oilseed rape embryos are provided as evidence against a weak acid trap mechanism acting in either the phloem mobility or the accumulation of glucosinolates in oilseed rape embryos. The phloem mobility of glucosinolates is explained by the intermediate permeability hypothesis. In conclusion, it would appear that glucosinolates like other groups of endogenous compounds have physicochemical properties allowing phloem mobility as predicted by the Kleier model.Keywords: Brassica napus, Ricinus communis, phloem mobility, glucosinolates, Kleier model.      

甘蓝型油菜BnCYP79B1基因的克隆与表达

硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1)。BnCYP79B1基因cDNA全长1 625bp,编码一个含有541个氨基酸、理论等电点为8.88。序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83%,推测蛋白氨基酸序列的相似性为99.26%。通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高。在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高。     

Secondary metabolites and plant/environment interactions: a view through Arabidopsis thaliana tinged glasses

Arabidopsis thaliana is a successful model plant for studying wide‐ranging topics including plant development, genetics and pathogen resistance. In addition, significant research has been conducted in the area of secondary metabolite biochemical genetics. The secondary metabolites in Arabidopsis include glucosinolates, terpenoids, phenylpropanoids, the alkaloid‐like camalexin, and other uncharacterized compounds. The genetic tools developed in studying secondary metabolite biochemistry are now being used to study how secondary metabolites control various biological processes. This includes compounds involved in plant/insect and plant/pathogen interactions, compounds preventing UV‐B damage, and compounds involved in hormone homeostasis. This review will describe what light Arabidopsis is shedding on the biological and ecological importance of specific secondary metabolites.     

Glucosinolates in the human diet. Bioavailability and implications for health

The glucosinolates are a large group of sulphur-containing glucosides found in brassica vegetables. After physical damage to the plant tissue, glucosinolates are broken down, by the endogenous enzyme myrosinase, releasing glucose and a complex variety of biologically active products. The most important and extensively studied of these compounds are the isothiocyanates. Glucosinolates can be degraded or leached from vegetable tissue during food processing, but thermal inactivation of myrosinase preserves some intact glucosinolates in cooked vegetables. Once ingested, any remaining intact glucosinolates may be broken down by plant myrosinase in the small intestine, or by bacterial myrosinase in the colon. Isothiocyanates are absorbed from the small bowel and colon, and the metabolites are detectable in human urine 2–3 h after consumption of brassica vegetables. Isothiocyanates are potent inducers of Phase II enzymes in vitro, and they have been shown to increase the metabolism and detoxification of chemical carcinogens in vitro and in animal models. Some of these compounds also inhibit mitosis and stimulate apoptosis in human tumour cells, in vitro and in vivo. This second effect raises the possibility that in addition to blocking DNA damage, isothiocyanates may selectively inhibit the growth of tumour cells even after initiation by chemical carcinogens. Epidemiological evidence supports the possibility that glucosinolate breakdown products derived from brassica vegetables may protect against human cancers, especially those of the gastrointestinal tract and lung. To define and exploit these potentially anticarcinogenic effects it is important to understand and manipulate glucosinolate chemistry and metabolism across the whole food-chain, from production and processing to consumption. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of glucobrassicin, epiprogoitrin and related breakdown products on locusts feeding: Schouwia purpurea and desert locust relationships

The glucosinolates of a Saharan crucifer Schouwia purpurea (Forskål) (Brassicaceae) were determined by liquid chromatography. Two of these glucosinolates and sinigrin were tested for their deterrent effect on Schistocerca gregaria (Forskål) (Orthoptera: Acrididae). Glucobrassicin, three indolyls and epigoitrin were synthesized for this purpose. Epiprogoitrin was extracted from Crambe seeds. Choice tests on artificial substrate compared feeding responses to glucosinolates and to related breakdown products released when the plant is eaten. Breakdown products were more efficient in deterring the generalist locust than were glucosinolates. Two patterns of dose responses were recorded: glucosidic compounds deterred or stimulated feeding, depending on the concentration tested; aglycones did not stimulate feeding at any concentration. Allyl isothiocyanate, a volatile compound, was a 100-fold higher deterrent than its substrate (sinigrin).     

Fungicidal Activity of Volatiles from Selected Cruciferous Plants against Resting Propagules of Soil-borne Fungal Pathogens

The efficacy of volatiles evolved from tissues of nine cruciferous plants against resting propagules of Fusarium oxysporum var radicis f. sp. lycopersici, Sclerotium cepivorum, and Sclerotinia sclerotiorum was tested. The cruciferous plants released biocidal compounds, mainly isothiocyanates, produced during the enzymatic degradation of glucosinolates present in the plant cells. Among the plants investigated, the highest fungicidal activity and also the highest concentration of isothiocyanates were found in Brassica juncea. The resting propagules of tested fungi differed significantly in their sensitivity towards volatiles released from plant tissues.     

Isolation of glucosinolates and the identification of o-(α-l-rhamnopyranosyloxy)benzylglucosinolate from Reseda odorata

A method has been developed for the quantitative isolation of glucosinolates by ion-exchange chromatography and high voltage electrophoresis avoiding strongly alkaline and acidic conditions. The compounds were identified by ¹H and ¹³C NMR spectroscopy and through the products arising from enzymatic, acid and alkaline hydrolysis. The method is well suited for the isolation and identification of glucosinolates containing aglucone parts which produce non-volatile compounds on enzymatic hydrolysis. The method has been used in the isolation and identification of 2-hydroxy-2-methylpropylglucosinolate from Reseda alba, 2-hydroxy-2-phenylethylglucosinolate from R. luteola and a new glucosinolate, o-(α-l-rhamnopyranosyloxy)benzylglucosinolate, occurring in R. odorata. The glucosinolate content in different parts of this plant has been determined and the metabolism of glucosinolates is briefly discussed.     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. II. Administration of Some Sulphur-35 and Carbon-14 Compounds and Localization of Metabolic Blocks

The aim of the investigation was to study the differences in the metabolism of substances that are utilized in the synthesis of glucosinolates between the Brassica napus cv. Bronowski, which is very low in glucosinolate content, and a cultivar (cv. Regina II) that contains approximately average amounts of these compounds. By experiments in which the plants were grown in nutrient solutions supplied with sulphate-³⁵S, it was shown that the rate of sulphate uptake was similar in the two cultivars. No accumulation of intermediate metabolites could be demonstrated by autoradiography in Bronowski. Sulphate-³⁵S, methionine-³⁵S, methionine-2-¹⁴C, 2-amino-6-(methylthio)caproic acid-2-¹⁴C, and S-(β-d -glucopyranosyl)-4-pentenethiohydroximic acid (desulpho-3-butenylglucosinolate) (glucose-U-¹⁴C or ³⁵S) were fed to shoots of the two cultivars. After incubation, the plant material was extracted with methanol. The extracts were separated into various fractions, and in some experiments glucosinolates or derivatives of their degradation products were isolated. When measuring the radioactivity of the various fractions or isolated products, the incorporation of radioactivity into glucosinolates was found to be poor in Bronowski from sulphate, methionine, and 2-amino-6-(methylthio)caproic acid. Desulpho-3-butenyl-glucosinolate was an efficient precursor of 3-butenylglucosinolate in Bronowski, but a poor precursor of 2-hydroxy-3-bute-nylglucosinolate, which suggests a metabolic block at the hydroxylation step in this cultivar. In Regina II desulpho-3-butenylglucosinolate was a good precursor of both 3-butenylglucosinolate and of 2-hydroxy-3-butenylglucosinolate, which demonstrates that these glucosinolates may be synthesized without prior formation of the corresponding co-methylthioalkyl glucosinolates and that the hydroxylation can take place after the formation of desulpho-3-butenylglucosinolate. The results indicate that the low glucosinolate content of Bronowski is caused by block(s) in the separate pathway leading to the biosynthesis of glucosinolates.     

Oviposition and chemosensory stimulation of the root flies Delia radicum and D. floralis in response to plants and leaf surface extracts from resistant and susceptible Brassica genotypes

In Brassica crops differences in susceptibility to root fly attack can be largely attributed to antixenotic resistance. Plants of four genotypes (two swedes and two kales) with widely differing resistance in field trials, were compared in laboratory choice assays for their susceptibility to oviposition by the root flies Delia radicum (L.) and D. floralis (Fallen) (Diptera, Anthomyiidae). For both species the preference among the genotypes corresponded to the susceptibility of the genotypes in the field. The preference ranking in response to surrogate leaves treated with methanolic surface extracts of the four genotypes was identical to the preference among potted plants, demonstrating that chemical factors on the leaf surface mediate host preference for oviposition in these species.For both species of fly, glucosinolates are major oviposition stimulants and for D. radicum an additional, nonglucosinolate oviposition stimulant, presently called CIF, is known. We describe a procedure for chromatographic separation of glucosinolates from CIF in leaf surface extracts. In oviposition-choice assays with D. radicum, the CIF-fractions of the two swede genotypes applied to surrogate leaves received a 1.8 and 4.6 times higher proportion of eggs than the respective glucosinolate-fractions, confirming the major importance of CIF as an oviposition stimulant. The genotype of swede that was preferred by both fly species in tests with plants and methanolic leaf surface extracts, also stimulated oviposition more in tests with the glucosinolate-fractions or the CIF-fractions derived from the surface extracts, respectively. Thus, glucosinolates and CIF together account for the observed preference among the genotypes and may also be responsible for their susceptibility under field conditions. In the two kale genotypes the preference for plants or surface extracts differed from the preference among the corresponding glucosinolate- and CIF-fractions, indicating that additional, as yet unknown chemical factors may also be involved.For both groups of stimulants tarsal chemoreceptors allow electrophysiological monitoring of glucosinolate- and CIF-activity in fractionated surface extracts. For D. radicum the chemosensory activity of both glucosinolate- and CIF-fractions corresponded to the respective behavioural activity in the oviposition preference tests, suggesting that preference for oviposition among genotypes can be predicted from the electrophysiological activity of their fractions. The chemosensory response of D. floralis, in particular to the CIF-fractions, was less pronounced than the response of D. radicum, indicating interspecific differences in the perception of the major oviposition stimulants. We discuss the potential application of electrophysiological techniques in support of other screening methods used in breeding for root fly resistance in Brassica crops.     

Hydrolysis products of glucosinolates in Brassica napus tissues as inhibitors of seed germination

Enzymatic hydrolysis of glucosinolates, a class of compounds found in Brassica species, results in a number of products with potential to inhibit seed germination. To investigate the impact of both volatile and water-soluble allelochemicals, germination bioassays using Lactuca sativa seeds were conducted with root and combined leaf and stem tissues of Brassica napus. Tissues in which glucosinolates were hydrolyzed to remove volatile glucosinolate degradation products were compared with intact tissues and water controls. Only tissues containing glucosinolates produced volatiles that inhibited germination. Volatiles were trapped and identified using GC-MS. Volatiles produced in greater quanitity from intact tissues than from tissues without glucosinolates were almost exclusively glucosinolate hydrolysis products. Water-soluble components also inhibited germination. Chemical analysis of extracts confirmed the presence of glucosinolate hydrolysis products, but indicated the involvement of additional allelochemicals, especially in leaf and stem tissues. Results support the proposal that glucosinolate-containing plant tissues may contribute to reductions in synthetic pesticide use if weed seeds are targeted.Abbreviations ITC isothiocyanates - CN organic cyanides - OZT oxazolidinethione - iRoot intact root tissue - iL&S intact leaf and stem tissue - hRoot hydrolyzed root tissue - hL&S hydrolyzed leaf and stem tissue     

Cloning and functional expression of two plant thiol methyltransferases: a new class of enzymes involved in the biosynthesis of sulfur volatiles

Glucosinolates are defensive compounds found in several plant families. We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles. In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an ArabidopsisEST showing high sequence homology to one TMT isoform. The genomic clone of cTMT1 was subsequently amplified by PCR. Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca. 25 kDa), but differing in 13 residues. The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O-or S-methyltransferases. A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs. Southern analysis indicated single copy for each TMT gene. The two cDNAs were expressed in Escherichia coli. The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme. These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs. The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates. The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.