Glucosinolates Metabolism and Occurrence in Vascular Plants

Glucosinolates (GS) are natural secondary plant metabolites that derive from amino acids (Halkier and Gershenzon 2006). The general chemical structure of GS was first proposed at the end of the nineteenth century by Gadamer, and was corrected in 1956 by Ettlinger and Lundeen (1956). This general structure of GS is characterised by a -C°N group, a sulfate and a b-D-glucopyranosyl. GS can be classified according to their precursor amino acid and the chemical structure and modifications of the side chain (-R); aliphatic, aromatic and indolyl GS can be distinguished (Fig. 7.1).

The widely accepted model for GS biosynthesis involves three major steps: side chain elongation, glucone biosynthesis, and side chain modification (Fig. 7.2). The presence of GS in Arabidopsis thaliana proved very useful when attempting to clarify the biosynthetic pathway of GS (Mikkelsen et al. 2002). GS biosynthesis starts with the hydroxylation of amino acids, followed by their decarboxylation to form an aldoxime. Alanine, methionine, valine and leucine are the precursors for the aliphatic GS, while the aromatic GS derive from phenylalanine and tyrosine. Tryptophan is the source of the indolyl GS. Cytochromes P450 belonging to the CYP79 family catalyse the conversion of the amino acids to aldoximes (Wittstock and Halkier 2002). Two members of another cytochome P450 family, CYP83A1 and CYP83B1, have been identified in Arabidopsis as the aldoxime-metabolising enzymes involved in the fast oxidation of the aldoximes and their conjugation with a sulphur donor, possibly cysteine. The thiohydroximic acid released is glucosylated (by uridine diphosphate

Fig. 7.1 General structure of the glucosinolates

CYP79

Cys UDPG PAPS

CYP79

Desulfoglucosinolatee

Glucosinolatee

Isothiocyanates

Epithionitriles

Oxazolidine-2-thiones

Thiocyanates

Alkyl-thiohydroxamate Thiohydroximic acic

Isothiocyanates

Epithionitriles

Oxazolidine-2-thiones

Thiocyanates

Nitriles

Unstable aglucon

Nitriles

Desulfoglucosinolatee

Glucosinolatee

Unstable aglucon

Myrosinase

Glucosinolate

Glucose

Fig. 7.2 Pathways for glucosinolate metabolism

glucose; UDPG) and sulfated (by 3-phosphoadenosine-5-phosphosulfate; PAPS) to form the GS core structure (Fig. 7.2). Several possible modifications of the R group determine the direction of GS hydrolysis and the activity of the products of hydrolysis (Halkier and Gershenzon 2006). Indeed, plant GS metabolism is regulated at multiple levels (genetic, environmental, transcriptional and metabolic).

Plants with GS also contain the enzyme b-thioglucoside hydrolase (commonly called myrosinase), which mediates GS hydrolysis. This enzyme is separated from its substrate in intact tissue, but when the tissue is damaged, the loss of cellular integrity results in the mixing of the enzyme and GS, causing the immediate hydrolysis of GS into an unstable aglycone. The aglycone rearranges spontaneously to form different products, such as isothiocyanates, oxazolidine-2-thiones, nitriles, epithi-onitriles and thiocyanates (Fig. 7.2). Myrosinase is highly specific, as it only uses GS as substrate and has no activity toward any O-glycosides or S-glycosides. Among the different types of GS, the myrosinase substrate range is variable, and some are highly specific. Myrosinase is encoded by a multigene family, and whereas Arabidopsis has four functional myrosinase genes (Xu et al. 2004), Brassica napus and Sinapis alba have 20 or more (Rask et al. 2000).

While GS are biologically inert, some of the products of their hydrolysis have important biological effects. Their nature depends mainly on the structure of the side chain, and also on the presence of epithiospecifier protein (ESP), the pH and the Fe2+ concentration of the medium. Usually, rearrangement of the aglycone at neutral pH will result in the formation of an isothiocyanate, while at acidic pH the nitrile derivative is the dominant product. This enzyme system has a broad pH range (Heaney and Fenwich 1993). Following the myrosinase hydrolysis of GS, nitriles and epithionitriles are generated by ESP, whereas isothiocyanates are generated in the absence of ESP (Chen and Andreasson 2001). Investigations with A. thaliana plants that overexpress ESP clearly support the view that isothiocyanates are more effective defences against herbivores than nitriles (Burrow et al. 2006).

Current research interest is focused mainly on the toxic and beneficial effects of plant GS in animal and humans. Antinutritional and toxic effects are of special relevance in some GS-rich fodder species, like rapeseed meal (Tripathi and Mishra 2007). Nitriles appear to be the main antinutritional factor responsible for growth depression in cattle. However, the most common GS hydrolysis products, isothiocyanates, are considered to be responsible for the protective and anticarcinogenic effects of a cruciferous-rich diet in humans. Their antifungal, antimicrobial, allelochemical and insecticidal properties contribute to the plant's defence mechanisms. While GS can defend plants against general pathogens and herbivores, they can also act as an attractant to GS-adapted specialists. For more than 30 years, GS have gained agricultural significance through the use of biofumigation (incorporation of harvested material into agricultural soil to suppress pathogens, nematodes and weeds).

A significant proportion of metal hyperaccumulators are members of Brassicaceae that typically contain GS. More than 80% of the identified GS occur in this plant family, which contains close to 3,000 species. However, GS are not restricted to Brassicaceae, as they occur throughout the order Capparales and even in Drypetes, a genus classically included in the Euphorbiaceae (De Craene and Haston 2006;

Rodman et al. 1996). At least 500 species from 15 other families of dicotyledonous angiosperms have been reported to contain one or more of the over 120 known natural GS. Some examples are Capparaceae, Tropaeolaceae, Moringaceae, Arabidaceae, Resedaceae and Euphorbiaceae.

Among the Brassicaeae, the genus Brassica contains a large number of commonly consumed vegetable species (cabbage, broccoli, radish, turnip), condiments (mustard, wasabi), oilseeds (canola, rapeseed), and forage crops (kale, forage rape). GS that have been found in all parts of these plants contribute significantly to their typical flavours. While more than 15 different GS can be found in the same plant, three to four usually predominate (Holst and Fenwick 2003). One of the best-characterised examples is the model plant A. thaliana, where up to 23 different GS were initially identified in the leaves and seeds (Hogge et al. 1988), with nine additional GS identified later (Wittstock et al. 2002).

The GS content in plants is highly variable and can range from less than 1% of the dry weight in some tissues of Brassica vegetables (Rosa et al. 1997) to 10% in the seeds of some plants, where GS may represent half of the sulfur content of the seeds. Occurrence and concentrations vary according to species, cultivar, tissue type, age, health and nutrition. Environmental factors (such as soil fertility and pathogen challenge) and plant growth regulators can affect GS levels and distribution among plant organs (Fahey et al. 2001). The accumulation of GS can also be induced after treatment with salicylic and jasmonic acids (Ludwing-Müller et al. 1997). Thus, the GS have diverse functions in plants and for plants, some of which may not have been discovered yet.

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