Glucosinolates and their degradation products

Glucosinolates and their Degradation Products

RICHARD

F. M I T H E N

School of Biosciences, Universityof Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK

1. II.

III.

IV.

V. VI.

VII.

I n t r o d u c t i o n ................................................................................................. S t r u c t u r e a n d B i o c h e m i c a l Diversity ........................................................ A. V a r i a t i o n in C h a i n Structure ............................................................. B. D i s t r i b u t i o n a n d E v o l u t i o n ................................................................ C. Diversity of Glucosinolates in Cruciferous Crops ........................... Biosynthesis ................................................................................................. A. G l u c o n e Biosynthesis .......................................................................... B. C h a i n E l o n g a t i o n : Biochemistry ....................................................... C. C h a i n E l o n g a t i o n : M o l e c u l a r G e n e t i c s a n d the I d e n t i f i c a t i o n of M A M Synthases .................................................................................. D. Side-chain Modification ..................................................................... E. Sites of Biosynthesis ............................................................................ G e n e t i c Analysis in B r a s s i c a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. M e n d e l i a n G e n e s R e g u l a t i n g C h a i n S t r u c t u r e in B r a s s i c a . . . . . . . . . . . . B. T h e D e v e l o p m e n t of Low-glucosinolate Oilseed R a p e : T h e Bronowski Block a n d Q T L M a p p i n g ................................................ C. E n h a n c i n g G l u c o s i n o l a t e s in Broccoli .............................................. D. G e n e t i c M o d i f i c a t i o n of Glucosinolates .......................................... E n v i r o n m e n t a l Factors Affecting G l u c o s i n o l a t e Expression ................ Glucosinolate D e g r a d a t i o n ....................................................................... A. Myrosinases .......................................................................................... B. Cellular a n d Subcellular L o c a t i o n of G l u c o s i n o l a t e s a n d Myrosinases .......................................................................................... C. P r o d u c t s ................................................................................................ D. M e t a b o l i s m a n d Detoxification of I s o t h i o c y a n a t e s ........................ Biological Activity of G l u c o s i n o l a t e s a n d t h e i r D e g r a d a t i o n Products ....................................................................................................... A. P l a n t - A n i m a l I n t e r a c t i o n s ................................................................. B. P l a n t - P a t h o g e n I n t e r a c t i o n s .............................................................. C. Flavour .................................................................................................. D. A n t i n u t r i t i o n a l Effects in Livestock a n d H u m a n s .......................... E. A n t i c a r c i n o g e n i c Activity ...................................................................

Advances in Botanical Research Vol. 35 incorporating Advances in Plant Pathology ISBN 0-12-005936-3

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Copyright © 2001 Academic Press All rights of reproduction in any form reserved

214

R.F. MITHEN

VIII. Conclusions.................................................................................................. 250 Acknowledgements..................................................................................... 250 References ................................................................................................... 250

Glucosinolates are the major class of secondary metabolites found in cruciferous crops. Following tissue damage they degrade to a variety of compounds of which isothiocyanates ("mustard oils") are the most prominent. These products are of considerable biological importance. They mediate plant-herbivore interactions, and have both positive and negative nutritional attributes. This review provides an overview of the biochemistry, genetic regulation and biological activity of glucosinolates and their degradation products. Emphasis is given to the considerable contribution that molecular genetic studies in Arabidopsis thaliana is providing to our understanding of fundamental aspects of the biosynthesis of these compounds, and to recent interest in the anticarcinogenic activity of isothiocyanates.

I.

INTRODUCTION

Glucosinolates are the main class of secondary metabolites found in cruciferous crops. Their presence is made known to us whenever we eat cruciferous vegetable and salad crops as they degrade immediately upon tissue damage due to the action of endogenous thioglucosidases ("myrosinases"), to release an array or products, of which isothiocyanates ("mustard oil") are the best known. These compounds are largely responsible for the characteristic hot and pungent flavours of crucifers. This review starts by considering the structure and biochemical diversity of glucosinolates, their biosynthesis from amino-acid precursors and their evolutionary origin. Several examples are provided of how our understanding of the biosynthetic pathway is advancing rapidly at present due to the contribution that molecular genetic studies with Arabidopsis thaliana is making to elucidating parts of the pathway that have proved intractable via a purely biochemical approach. The diversity of chain structures is explained as an interaction between genes governing aminoacid elongation and those determining chain modification. The genetic regulation of glucosinolate accumulation in Brassica is considered, with some discussion of how comparative genetic studies with A. thaliana may help to resolve the nature of quantitative trait loci that regulate glucosinolate accumulation. The review then considers the degradation of glucosinolates via myrosinases, to isothiocyanates and other products. The biological activities of these compounds are discussed in the context of the manner by which they mediate plant-herbivore interactions, their antinutritional effects when consumed mainly by livestock and, finally, their anticarcinogenic activity.

GLUCOSINOLATES AND T H E I R D E G R A D A T I O N PRODUCTS

215

Analytical methods for glucosinolates and their hydrolytic products are not reviewed. Verkerk et al. (1998) provide a useful overview of different methods of analysis of glucosinolates. In general, high-performance liquid chromatography (HPLC) analysis of desulphoglucosinolates using similar methods to that described by Heaney and Fenwick (1993) is the most widely adopted technique. This method is readily coupled to mass spectrometry via atmosphere pressure chemical ionization (Ishita et al., 1997), which has greatly aided identification of desulphoglucosinolates with similar retention times. A variety of methods are required for the analysis of glucosinolate degradation products, owing to their diverse structures resulting in contrasting physical and chemical properties. Volatile isothiocyanates and many nitriles are readily analysed by gas chromatography-mass spectrometry (GC-MS) (Spencer and Daxenbichler, 1980), whereas oxazolidinethiones (from 3-hydroxyalkenyl glucosinolates) can be analysed by HPLC (Quinsac et al., 1992). Non-volatile methylsulphinylalkyl isothiocyanates, of interest due to their anticarcinogenic properties, can be analysed by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization, as can N-acetyicysteine and other thiolisothiocyanate conjugates (Rose et al., 2000). II.

STRUCTURE AND BIOCHEMICAL DIVERSITY A.

VARIATION IN CHAIN S T R U C T U R E

The glucosinolate molecule consists of a 3-thioglucose moiety, a sulphonated oxime moiety and a variable side chain, derived from an amino acid (Fig. 1). This structure was first proposed by Ettlinger and Lundeen (1956), and confirmed by subsequent synthesis (Ettlinger and Lundeen, 1957) and X-ray crystallography (Waser and Watson, 1963). Glucosinolates with more than 115 side-chain structures have been described (Fig. 2). Only seven of these side-chain structures correspond directly to a protein amino acid (alanine, valine, leucine, isoleucine, phenylalanine, tyrosine and tryptophan). The remaining glucosinolates have side-chain structures that arise in three ways. Firstly, many glucosinolates are derived from chain-elongated forms of protein amino acids, notably from methionine, but also from phenylalanine and branch/ S - - ~--Glucose R--C

II ~"oso~-

Fig. 1. Structure of glucosinolates. R represents the side chain.

216

R.F. MITHEN CH3~ CH3"~[CH2] n

Methyr Alkyl (n= 1-6)

CH3~S--[CH2] n

Methylthioalkyl (n = 2-10)

CH3~I--[CH2]n

Methylsulphinylalkyl (n = 3-11)

O O II CH3~I--[CH2]n

Methylsulphonylalkyl (n = 3-10)

O CH3~S--CH=CH--[CH2]n

4-Methylth Jo-3-butenyl

C H 3 ~ S--- C H ~ CI-F~ [CH2] n II O

4-Methylsulphinyl-3-butenyl

O II C H 3 - - - - S - - C H : CH--[CH2] n II O C H 3 ~ [C H2n]-h'~i~,----[C H 2 ] n ~

4-Methylsulphonyl-3-butenyl

Oxylalkyl (n = 1-2; rf = 1-2)

O C H 2 ~ CH-~[CH2] n CH~

CI-I~CH--CH2~

I

Alkenyl (n = 1-4) 2-Hydroxy-3-butenyl

OH

CH~

CH--CH2~CH--CH2----

I

2-Hydroxy-4-pentenyl

OH CH2--[CH2]n I OH

Hydroxyalkyl (n = 2-3)

CH3---?I-I~[CH2]n

Hydroxylakyl (n = 1-2)

/

OH

Benzoyloxyalkyl (n = 2-6) O

Fig. 2.

Examples of side-chain structures of glucosinolates.

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

CH3~CH-I CH3 CH3---CH2--CH~ I CH3 CH3---~H--[CH2]n CH3

Ho__

Methylethyl

1-Methypropyl

Methylalkyl(n = 1-4)

4-Hydroxyphenyl

3-Methoxy-4-hydroxyphenyl CH30

3,4-Dimethoxyphenyl CH30

~

-CH2~

HO~~CH2~

~

Benzyl

4-Hydroxybenzyl

CH2"~

3-Methoxybenzyl

CH30 HO~~CH2-HO Fig. 2.

Contd.

3,4-Dihydroxybenzyl

217

218

R. F. MITHEN

~

-IcF--

Benzoyl

O

~

-[CH2ln

Phenylalkyl (n = 2-4)

2-hyd roxy-2-phenylethyl OH

~

CH~

3-1ndolylmethyl

I

H

OH

/~~CH2-'--

4-Hydroxy-3-indolylmethyl

I

H

~

OOHs CH2----

4-Methoxy-3-indoymethyl

~-CH2--

1-Methoxy-3-indolylmethyl

I H

I

OCH 3

~--CH2---I

SO@Fig. 2.

Contd.

1-Sulphonate-3-indolylmethyl

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

219

chain amino acids. Secondly, the structure of the side chain may be modified after amino-acid elongation and glucosinolate biosynthesis by, for example, the oxidation of the methionine sulphur to sulphinyl and sulphonyl, and by the subsequent loss of the w-methylsulphinyl group to produce a terminal double bond. Subsequent modifications may also involve hydroxylation and methoxylation of the side chain. Chain elongation and modification interact to result in several homologous series of glucosinolates, such as those with methylthioalkyl side chains ranging from CH3S (CH2) 3 to CH3(CH2)8, and methylsulphinylalkyl side chains ranging from C H 3 S O ( C H 2 ) 3 t o CH3SO(CH2)11. In this review, I adopt the terminology of 3C, 4C, 5C .... , nC glucosinolates to refer to methionine-derived glucosinolates, which are based upon a propyl, butyl, pentyl, ..., n-alkyl side chains as a result of the addition of one, two, three ..... ( n - 2) methylene groups to methionine. Thirdly, some glucosinolates occur that contain relatively complex side chains, such as o-(a-L-rhamnopyranosyloxy)-benzyl glucosinolate in Reseda odorata (Olsen and S0rensen, 1979) and glucosinolates containing a sinapoyl moiety in Raphanus sativus (Linscheid et al., 1980). Of these 115 or so glucosinolates, approximately 50% arc derived from chain-elongated methionine. A further 10% are each derived from tryptophan (indolyl side chains), phenylalanine and/or tyrosine (aromatic side chains) and from elongated forms of phenylalanine. Most of the remaining glucosinolates are probably derived from branch-chain amino acids, alanine or methionine, although the amino-acid precursors of some are difficult to speculate upon (Fig. 2).

B.

DISTRIBUTION AND EVOLUTION

Glucosinolates are found in 16 dicotyledonous plant families. Molecular phylogenetic studies based upon DNA sequencing of the chloroplast rbcL gene and the nuclear 18S ribosomal R N A gene, combined with morphological and biochemical analysis, suggests that all glucosinolatecontaining taxa, with the exception of the genus Drypetes of the Euphorbiaceae, have a monophyletic origin and can be included in an expanded Capparles (syn. Brassicales) (Rodman et al., 1996, 1998; Angiosperm Phylogeny Group, 1998). Thus, it is likely that the glucosinolate-myrosinase system arose independently on two occasions, with one of these origins giving rise to all but one of extant glucosinolate containing taxa. Matching the revised phylogeny of the Brassicales against the amino-acid precursors of glucosinolates within the different families reveals an intriguing pattern. While all families have at least some representatives, which contain glucosinolates derived from phenylalanine and/or tyrosine

220

R.F. MITHEN Amino acid precursor

Alanine

_ _

Capparis Cleome Arabidopsis --] Brassica Reseda

Elongated methionine- Elongatedphenylalanine

'--- Gyrostemon Tovaria Tryptophan

E -

I

E

Branch-chain amfno acids Tyrosine Phenylalanine

Pentadiplandra Koeberlinia Batis

-

Salvadora Setchellanthus

Limnanthes~] E

FIoerkea .__1 Carica

--- Moringa Tropaelum

Bretschneidera Akania ~

Capparaceae 45/800 Brassicaceae 350/3000 Resedaceae 6/70 Gyrosyemonaceae 5/17 Tovariaceae 1/2 Pentadiplandraceae 1/1 Koeberliniaceae 1/1 Bataceae 1/2 Sa[vadoraceae 3/12 Capparaceae Limnanthaceae 2/11 Cariceae 4/30 Moringaceae 1/10 Tropaeolaceae 3/92 Bretschneideraceae 1/1 Akaniaceae 1/1

Gossypium Bombax Acer Cupaniopsis Coleonema

Fig. 3. Phylogenetic relationships of glucosinotate-containing plant families (bold lines) determined by the DNA sequence of the chloroplast rbcL gene and the nuclear 18S ribosomal RNA gene, based upon Rodman et al. (1998), where further details are provided, and amino-acid precursors for glucosinolate synthesis. This scheme is only provisional as the glucosinolate content of many taxa remains to be thoroughly investigated.

(e.g. benzyl and hydroxybenzyl glucosinolate), and probably from branchchain amino acids (e.g. isopropyl and sec-butyl glucosinolates), considerably greater biochemical diversity is found within the families of more recent evolutionary origin. Thus, the emergence of new taxa is associated with the use of novel amino-acid precursors for glucosinolates biosynthesis (Fig. 3). The Brassicaceae and Capparaceae are by far the largest families within the Brassicales, and contain the greatest number and diversity of glucosinolates. A survey of glucosinolate content of 297 wild species is reported by Daxenbichler et al. (1991). Aspects of glucosinolate biochemistry suggest an evolutionary relationship with cyanogenic glycosides. Both classes of secondary metabolites are derived

GLUCOSINOLATES AND THEIR D E G R A D A T I O N PRODUCTS

221

from tyrosine/phenylalanine and branch chain amino acids, and have oximes as intermediates (see below). Cyanogenic glycosides occur widely in flowering plants and non-flowering plants, suggesting an early evolutionary origin. In contrast, glucosinolates have a restricted occurrence in a relatively small number of plant families (see above). It is likely that glucosinolates may have arisen through the diversion of the oxime away from cyanogenic glycosides biosynthesis via conjugation with cysteine. The resultant highly reactive thiohydroximate would be detoxified by first conjugation with sulphate and then with glucose, resulting in the glucosinolate molecule. C.

DIVERSITY OF GLUCOSINOLATES IN C R U C I F E R O U S CROPS

The major cruciferous crops have a restricted range of glucosinolates. Fenwick et al. (1983) provides a useful summary of glucosinolate content in the major crop species. All of these tend to have a mixture of indolylmethyl and N-methoxyindolylmethyl glucosinolates and either a small number of methionine-derived or aromatic glucosinolates. Brassica nigra and B. carinata contain only 2-propenyl glucosinolate, while B. juncea has either 2-propenyl or a mixture of 2-propenyl and 3-butenyl glucosinolate. B. rapa and B. napus lack 3C glucosinolates and contain a mixture of 3butenyl and 4-pentenyl glucosinolates and their hydroxylated homologues. The greatest diversity is observed within B. oleracea. Genotypes of this species have either 3C or 4C glucosinolates. Broccoli (B. oleracea vat. italica) accumulates 3-methylsulphinylpropyl and 4-methylsulphinylbutyl glucosinolates, while other botanical forms of B. oleracea have mixtures of 2-propenyl, 3-butenyl and 2-hydroxy-3-butenyl. Some cultivars of cabbage and Brussels sprouts also contain significant amounts of methylthioalkyl glucosinolates. Several surveys of glucosinolate variation between cultivars of Brassica species have been reported, for example, B. rapa (Carlson et al., 1987a; Hill et al., 1987) and B. oleracea (Carlson et al., 1987b; Kushad et al., 1999). In contrast to Brassica, Sinapis alba accumulates mainlyp-hydroxybenzyl glucosinolate. The distinctive taste of many minor horticultural cruciferous crops is due to their glucosinolate content. For example, watercress accumulates large amounts of phenylethyl glucosinolate, rockets (Eruca and Diplotaxis species) contain 4-methylthiobutyl glucosinolate, and cress (Lepidium sativa) contains benzyl glucosinolate. Glucosinolates in the seeds of minor oil crops may become of greater importance if these crops are more widely exploited. For example, Camelina sativa accumulates 9-methylsulphinylnonyl, 10-methysulphinyldecyl and ll-methylsulphinylundecyl glucosinolates (Daxenbichler et al., 1991). Limnanthes species, possible sources of industrially fatty acids, accumulate substituted aromatic glucosinolates (Bartelt and Mikolajczak, 1989).

222

R.F. MITHEN

III.

BIOSYNTHESIS

The biosynthesis of glucosinolates can be considered in three parts: chain elongation of precursor amino acids, the synthesis of the glucone moiety, and chain modification (Fig. 4). A. GLUCONEBIOSYNTHESIS The first step in glucosinolate biosynthesis is the conversion of the amino acid to an oxime (Fig. 5). The mechanism by which this occurs has been rather controversial. Biochemical studies have suggested that several different types of enzymes convert the different amino acids to oximes, while more recent studies with A. thaliana suggest that amino acid-oxime conversions may be catalysed by cytochrome P450 monooxygenases of the CYP79 family. The best characterized system is the conversion of tyrosine and phenylalanine to their corresponding oximes ( D u e t al., 1995; Bak et al., 1999), as precursors of benzyl and hydroxybenzyl glucosinolates. The involvement of cytochrome P450s in the conversion of these amino acids has been elegantly demonstrated in a series of studies by Halkier and colleagues, and builds upon a comparison of the biosynthesis of cyanogenic Amino acid

Chain elongation (Fig. 6) Elongated amino acid

Glucosinolate biosynthesis (Fig. 5) Glucosinolate

Oxidation, desaturation, hydroxylation, etc. (Fig. 7) Chain modification

Myrosinase and cofactors mediated hydrolysis (Plate 3 and Fig. 9)) Degradation products, isothiocyanates, nitriles, etc.

Mammalian metabolism of isothiocyanates (Fig. 10) N-Acetylcysteine-isothiocyanate conjugates

Fig. 4. Summary of metabolic pathways for glucosinolate synthesis and degradation.

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

@

223

R~CH--COOH

I

NH 2

Amino acid

Elongation (Fig. 6)

R~CH2--C / II

Oxime

N...OH H R~CH2--C / II N+

AcFnitro compounds

~ O-/ \OH

+ cysteine

S--CH2--CH--COOH

R~CH2__C /

I

II N

NH2

\OH

S-Alkylthiohydroximate

SH R--CH2--C / II UDPG

~

Thiohydroximate

N\OH S--Glucose

R--CH2--C / II N\

PAPS

~

Desulphoglucosinlate

OH S--Glucose

R~CH2--C / II

~

Glucosinolate

N\OSO3-

Chain modification

Fig. 5. Glucosinolate biosynthesis. The central steps involving the conversion of the oxime to a thiohydroximate are speculative. glycosides to glucosinolates (Bak et al., 1998; Kahn et al., 1999). The cytochrome P450 CYP79A1 catalyses the conversion of tyrosine to phydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glycoside dhurrin in sorghum. A. thaliana does not normally express benzyl glucosinolate, although trace levels can sometimes be found (Wittstock and

224

R.F. MITHEN

Halkier, 2000). However, when CYP79A1 was expressed in this species, benzyl glucosinolate was detected in surprisingly large amounts in leaf tissue (Bak et al., 1999). This is explained by the p-hydroxyphenylacetaldoxime being channeled into benzyl glucosinolate biosynthesis due to the expression of endogenous glucosinolate genes with low substrate specificity, as opposed to cyanogenic glycosides, which A. thaliana does not synthesize. A similar cytochrome P450, CYP79A2, was cloned from A. thaliana itself. This enzyme catalyses the conversion of phenylalanine, as opposed to tyrosine, into phenylacetaldoxime. When constitutively expressed, benzyl glucosinolate was again found in A. thaliana (Wittstock and Halkier, 2000). CYP79A2 lacked any substrate specificity for other amino acids, which act as glucosinolate precursors, such as L-tryptophan, Lmethionine, DL-homo-phenylalanine, indicating the high substrate specificity of this part of glucosinolate biosynthesis, which contrasts to latter steps (see below). Indolyl glucosinolate biosynthesis requires the conversion of trytophan to indole-3-acetaldoxime, which is also thought to be a precursor in the synthesis of IAA. Biochemical studies have suggested that this conversion is mediated by plasma membrane-bound peroxidases (Ludwig-Mtiller and Hilgenberg, 1988; Ludwig-Mtiller et al., 1990), and it has been assumed that these enzymes are also involved in indolyl glucosinolate biosynthesis (Bennett et al., 1996). However, recent studies withA, thaliana suggest that the conversion of tryptophan to its oxime is catalysed by the cytochrome P450s, CYP79B2 and CYP79B3 (Hull et al., 2000). Disruption of these genes in A. thaliana will lead to their role in glucosinolate biosynthesis being confirmed. Interestingly, CYP79B2 and CYP79B3 are similar to CYP79A2 in that they only possess a single intron. Other members of the CYP79 family have one or more additional introns. Glucosinolates from chain-elongated methionine and phenylalanine are of particular significance in economically important Brassica crops owing to the biological activity of their hydrolytic products. Biochemical studies in Brassica with isolated microsomes have reported that the conversion of these amino acids to their corresponding oximes is catalysed by at least two flavin-containing monooxygenases. Firstly, one with substrate specificity for homophenylalanine, the precursor of phenylethyl glucosinolate and, secondly, one with specificity for di- and tri-homomethionine, the precursors for aliphatic glucosinolates with 4C and 5C side chains, such as 3-butenyl and 4-pentenyl glucosinolates (Bennett et al., 1993, 1995, 1996; Dawson et al., 1993). Somewhat surprisingly, a flavin-containing monooxygenase was not found that had specificity for homomethionine, the precursor of 3C glucosinolates, such as 2-propenyl glucosinolate. It likely that further molecular genetic studies with A. thaliana will confirm whether these enzymes are involved in oxime synthesis or whether enzymes encoded by further members of the CYP79 gene family are involved.

GLUCOSINOLATES AND T H E I R D E G R A D A T I O N PRODUCTS

225

The intermediates between the oximes and the thiohydoximates have not been identified (Fig. 5) and this part of the pathway must be considered speculative. It has been proposed that the oxime is oxidized to an aci-nitro compound (Ettlinger and Kjaer, 1968). This conjugates with cysteine, which probably functions as the thiol donor (Wetter and Chisholm, 1968). This reaction may be catalysed by a glutathione-Stransferase. The resulting S-alkylthiohydroximate may be cleaved by a CS-lyase to yield the thiohydroximate, although experimental evidence is lacking. As with resolving other parts in the pathway, it is likely that studies with A. thaliana will provide an answer to this crucial step in glucosinolate biosynthesis. The final steps in the biosynthesis of the glucone are the most fully understood. The thiohydroximate is S-glucosylated by a soluble UDPG:thiohydroximate glucosyltransferase (S-GT) to produce a desulphoglucosinolate (Matsuo and Underhill, 1969; Reed et al., 1993; Guo and Poulton, 1994; GrootWassink et al. 1994, Anonymous, 1997). This is sulphated by a soluble 3'-phosphoadenosine 5'-phosphosulphate (PAPS): desulphoglucosinolate sulphotransferase (Jain et aI., 1990a). Both of these enzymes have been partially purified from several species. In B. juncea, the enzymes involved in sequential glycosylation and sulphation co-purify, suggesting that they may exist as an enzyme complex (Jain et al., 1990b). However, similar results were not obtained when purified from A. thaliana (Guo and Poulton, 1994). Characterization of S-GT from B. oleracea has shown that it has high substrate affinity for thiohydroximates, but little specificity for side-chain structure (GrootWassink et al., 1994). Sulphotransferases have been purified from Lepidium (Glendenning and Poulton, 1990) and B. juncea (Jain et al., 1990a). As with S-GT, the enzymes were able to catalyse the sulphation of several different desulphoglucosinolates. S-GT genes have been cloned from Brassica (Anonymous, 1997), possibly facilitating genetic modification. B.

CHAIN ELONGATION: BIOCHEMISTRY

While some glucosinolates are synthesized from chain-elongated forms of valine and phenylalanine, by far the most are synthesized from elongated forms of methionine. These are restricted to the Brassicaceae and the Capparaceae (recent phylogenetic analyses have incorporated the Cappararaceae within an enlarged Brassicaceae (Angiosperm Phylogeny Group, 1998)). While methionine may be elongated with the addition of a single or up to nine methylene groups, most taxa with the Brassicaceae have only a restricted number of chain lengths, which can be divided into three classes. "Short chain", involving the addition of one, two, three and (rarely) four methylene groups to methionine, such as found in Brassica crops, "long

226

R.F. MITHEN TABLE I

Variation in chain length of methionine-derived glucosinates based on data from Daxenbichler et al. (1991)

Chain length Short chain Taxa

3C

4C

5C

A. thaliana B. nigra B. oleracea B. rapa B. napus Hesperis matronalis Lepidium spp. Rorippa nasturtiumaquaticum Arabis glabra Arabis hirsuta Arabis alpina Camelina sativa

++ ++

++

+

++

++ ++ ++

++ ++ ++

6C

Long chain

Very long chain

7C

8C

9C

+

++

++ ++

++

10C llC

+

++

++ ++

++ ++

++ ++

q-q-

+ +, abundant; +, trace amounts. chains" involving the addition of five or six, and "very long chain", involving the addition of seven, eight or nine methylene groups (Table I). A. thaliana is unusual as it combines both short-chain and long-chain methioninederived glucosinolates. Biochemical studies, involving the administering of 14C-labelled acetate and 14C-labelled amino acids and subsequent analysis of the labelled glucosinolates (Underhill et al., 1962; Chisholm and Wetter, 1964; Graser et al., 2000), suggests that amino-acid elongation is similar to that which occurs in the synthesis of leucine from 2-keto-3-methylbutanoic acid and acetyl CoA (Strassman and Ceci, 1963; Umbarger, 1997). The amino acid is transaminated to produce an ~-keto acid, followed by condensation with acetyl CoA, isomerization involving a shift in the hydroxyl group and oxidative decarboxylation to result in an elongated keto acid, which is transaminated to form the elongated amino acid (Fig. 6). It is likely that the elongated keto acid can undergo further condensations with acetyl CoA to result in multiple chain elongations. These are particularly characteristic of glucosinolates derived from methionine in which up to nine methylene groups may be added (Fig. 2 and Table I). Graser et al. (2000) provide evidence through the administration of [~SN]methionine that the transamination reactions and the keto-acid elongation reactions occur in the same subcellular compartment; two thirds of the labelled amino group, which was removed to form the keto acid in the first transamination reaction, was subsequently returned to the elongated keto acid during the second

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

227

R--CH2--CH--COOH

I

NH2 Transamination

R---CH2--O--COOH

II

/

Condensation

0

+

+ Acetyl CoA COOH

• ....

I

R---CH~--CHz~COOH OH further condensation reactions

Isomerization COOH

I

R---CH2~CH-- CH--COOH

I

OH Decarboxylation

R__CHz__CH2__CH__COOH . . . .

li

0

Transamination

FI~ CH2-- CH2----CH-- COOH

I

NH2 Glucosinolate biosynthesis (Fig. 5) 3C glucosinolates

; Chain modification

Fig. 6. Amino-acid chain elongation. transamination reactions to restore the amino acid. There has been relatively little attempt to purify enzymes from this part of the biosynthetic pathway. A potential methionine aminotransferase has been partially purified from B. carinata (Chapple et al., 1990), but its involvement in glucosinolate biosynthesis has not been established. C.

CHAIN ELONGATION: MOLECULAR GENETICS AND IDENTIFICATION OF MAM SYNTHASES

Molecular genetic studies in A. thaliana have provided an alternative to a purely biochemical approach to elucidating the regulation of chain

228

R.F. M1THEN

elongation. A. thaliana ecotypes vary in the length of their aliphatic side chain. Two major groups occur, which are independently regulated. Firstly, short-chain methionine-derived glucosinolates. These contain glucosinolates, which have undergone one, two or three rounds of elongation to produce glucosinolates with a 3C, 4C or 5C side chain. The relative proportions of these vary between ecotypes. For example, some ecotypes, such as Ler (Landsberg erecta), La-0, Es-0 and Can-0, have predominantly glucosinolates with a side chain derived from methionine, which has undergone one round of elongation, such as 3-hydroxypropyl, 3methylsulphinylpropyl, 2-propenyl and 3-benzoylopropyl glucosinolates (i.e. 3C glucosinolates), while other ecotypes, such as Col-0 (Colombia), Cvi-0, An-1 and Cen-0, have glucosinolates derived from methionine, which have undergone two rounds of elongation, such as 4-methylsulphinylbutyl, 3butenyl and 4-benzoylobutyl glucosinolate (i.e. 4C glucosinolates) (Mithen and Campos, 1996). The second group comprise long-chain methioninederived glucosinolates side chains based upon 5 and 6 rounds of elongation (e.g. 7-methylsulphinylheptyl and 8-methylsulphinoctyl glucosinolate). All A. thaliana ecotypes have similar amounts of these glucosinolates (unpublished). Haughn et al. (1991) described the isolation of six mutants of the ecotype Colombia with altered glucosinolate phenotype. Some of these clearly exhibited alterations in the ratio of short-chain glucosinolates, providing a useful resource for subsequent genetic studies. For example, the mutant TU1 exhibited an increase in 3C and 8C glucosinolates and a decrease in 4C glucosinolates, owing to a recessive allele, gsml, of a single nuclear gene. Campos de Quiros et al. (2000) adopted a different approach. Rather than using mutants, they demonstrated that the differences between the 3C and 4C glucosinolate phenotypes of L. erecta and Colombia was due to allelic variation at a single Mendelian locus on chromosome 5, which they designated G S L - E L O N G . Molecular genetic analysis of this locus revealed the presence of two adjacent genes, which have high homology to isopropylmalate synthase and homocitrate synthase, both of which catalyse the condensation between keto acids and acetyl CoA, similar to the second reaction of the proposed pathway of methionine elongation (Fig. 6). Thus, these genes can be termed MAM synthases (methylthioalkylmalate synthases). Functional analysis of these genes has confirmed their role in the synthesis of chain-elongated glucosinolates; alterations in expression either through transposon insertion induced knockouts or through transformation with antisense constructs alters the side-chain lengths of methionine-derived glucosinolates (G. Cardon, unpublished). Several genetic studies are consistent with there being three or four MAM synthase loci in A. thaliana and nine or ten in B. oleracea. At all of these loci, the MAM synthase catalyses the elongation of methionine, although to different extents. At some loci there is allelic variation. For example, in A. thaliana and B.

GLUCOSINOLATESANDTHEIRDEGRADATIONPRODUCTS Locus

229

Glucosinolates

MAM-1 Methionine

~--

--~.~allelic MAM-2

Methionine

[ ~

2-amino-5-methylthiopentanoic acid ~ variation

MAM-3 / Methionine - - l ~ l i c

MAM-4 Methionine

3C

3C

or

2-arnino-6-methylthiohexanoic acid ~

4C

2-amino-6-rnethylthiohexanoic acid ~

4C

variation

or

2-amino-7-methylthioheptanoic acid ~

5C

2-amino-9-methylthiononanoic acid

7C

and

MAM-5 Methionine

2-amino-5-methylthiopentanoic acid ~

~

2-amino-10-methylthiodecanoic acid

8C

2-amino-11-methyltNoundecanoic acid ~

9C

2-amino-12-methyithiododecanoic acid ~

10C

and and

2-amino-13-methylthiotridecanoic acid ~

11C

Fig. 7. Model of chain elongation in which a combination of null and functional alleles at several MAM synthase loci determine the synthesis of elongated forms of methionine, which form precursors of glucosinolates of different chain lengths. A. thaliana has three or four MAM loci (equivalent to MAM-1, MAM-2 and MAM-4). B. oleracea has about 10 loci, which include several copies of MAM-2.

oleracea, alternative alleles at one locus catalyses either one round (to make 3C) or two rounds (4C) of elongation, whereas at other loci a single allele may make a small number of elongated methionine homologues (Fig. 7). While these studies have concentrated on chain-elongated glucosinolates derived from methionine, the synthesis of phenylethyl glucosinolates from phenylalanine probably follows a similar pathway. Despite the elucidation of the importance of genes that catalyse condensation between keto acids and acetyl CoA, several questions remain concerning their substrate specificity and their role in both primary (i.e. leucine biosynthesis) and secondary (chain-elongated glucosinolates) metabolism. It is likely that the recent evolutionary origin of chain-elongated glucosinolates, particularly in the

230

R.F. M1THEN

Brassicaceae, is the result of the duplication and subsequent change in substrate specificity of isopropylmalate synthase, and its recruitment into an existing secondary metabolism pathway resulting in novel biochemical diversity. In addition to similar chain-elongation processes in primary metabolism, for example, in the synthesis of leucine and in the oxo-adipate pathway of lycine biosynthesis in fungi (Bhattacharjee, 1985), a similar pathway has also been described for the elongation of pyruvate to form long chain keto acid sucrose esters in Solanaceae (Kroumova et al., 1994), and in the synthesis of the alkyl portion of mercaptoheptanoic acid in Methanoccous jannaschii (Howell et al., 1998). D.

SIDE-CHAIN MODIFICATION

Glucosinolates derived from all amino acids may undergo modifications to their chain structure. This is likely to occur after the synthesis of the g!ucone moiety, either by the modification of the intact glucosinolate or the desulphoglucosinolate. As with other aspects of glucosinolates, most attention has been paid to methionine-derived glucosinolates. Following the biosynthesis of methylthioalkyl glucosinolates from methionine, the side chain may undergo various modifications. The suggested pathway involves an initial oxidation to methylsulphinylalkyl (and probably, in some species, methylsulphonylalkylglucosinolates), followed by the removal of the methylsulphinyl group and desaturation to result in alkenyl glucosinolates (Fig. 8). Alternatively, hydroxylation may result in 3-hydroxypropyl or 4hydroxybutyl glucosinolates (Giamoustaris and Mithen, 1996; Mithen et al., 1995a; Hall et al. 2000). Within this part of the pathway there is considerable scope for variation. For example, 4-methylsulphinylbutenyl glucosinolate, found exclusively in Raphanus, probably results from the desaturation of the corresponding methylsulphinylbutyl glucosinolate but without associated methylsulphinyltransferase activity. 3-Propyl and 4-butyl glucosinolates may also be derived by removal of the methylsulphinylgroup but without any further modifications. The alkenyl glucosinolates, 3-butenyl and 4-pentenyl may undergo ~3-hydroxylation, with important consequences for the nature of hydrolytic products (Fig. 8). The biochemistry underlying these modifications have received relatively little attention, except for the final hydroxylation of alkenyl glucosinolates, which may be catalysed by a cytochrome P450 hydroxylase (Rossiter and James, 1990; Rossiter et al., 1990). As with chain elongation, molecular genetic studies with A. thaliana are providing a means to elucidate the biochemical and genetic basis behind some of these modifications. In addition to variation in chain length, A. thaliana ecotypes vary in the types of chain modifications that are observed. Differences are usually more apparent when comparing glucosinolates in leaves rather than those in seeds, which are usually dominated by methylthioalkyl and benzoyl-

GLUCOSINOLATES ANDTHEIRDEGRADATION PRODUCTS

231

CH3--S--CH2--CH2--CH--COOH I NH2

I~Chainelongation

~ Glucosinolatebiosynthesis CH3--S--CH2--CH2--CH2--CH2--GSL 4-methylthiobutyl

~lGSL-OXID CH3--SO~CH2--CH2--CH2--CH2--GSL 4-methylsulphinylbutyl CH3--[CH2]3--GSL

A ~ ~ GSL-

CH3--SO~CHz CH--CH2--CH2--GSL 4-methylsulphinylbutenyl CH2z CH--CH2--CH2--GSL 3-butenyl

~ GSL-OH

Butyl ?H2-[CH2]3-GSL OH 4-hydroxy-butyl ~C---O---[CH2]4--GSL 4-benzoyloxybutyl

CH2z CH--CH--CH2--GSL I OH 2-hydroxy-3-butenyl Fig. 8. Chain modification of methionine-derived glucosinolates. This scheme is speculative. The precise pathway of the majority of these modifications are not known.

oxyalkyl glucosinolates. For example, in leaves of L. erecta, the most abundant glucosinolate is 3-hydroxypropyl glucosinolate, while in leaves of Colombia the most abundant glucosinolate is 4-methylsulphinylbutyl. Through genetic analysis of A. thaliana ecotypes, a locus (GSL-OHP), which regulates the conversion of methylsulphinylalkyl to hydroxyalkyl glucosinolates, has been positioned on chromosome 4. This locus is in the same interval between markers as a locus mapped in a separate cross, which regulates the conversion of methylsulphinylalkyl to alkenyl glucosinolates (GSL-ALK). Molecular genetic analysis has shown that within a 50 kb region, which spans this locus, a series of genes with homology to 2-oxogluturate-dependent dioxyengenases occur (Hall et al., 2000). Similar genes catalyse desaturation and hydroxylation reactions in several metabolic pathways (Prescott and Lloyd, 2000) and are

232

R.F. MITHEN

prime candidates for involvement in chain modification. However, other genes in this region may also be involved.A, thaliana ecotypes also vary in the extent of hydroxylation of 3-butenyl glucosinolate, although this gene has not yet been mapped. p-Hydroxybenzyl glucosinolate may arise in two ways, either derived directly from tyrosine, or from phenylalanine with subsequent hydroxylation. In Sinapis alba, the former route occurs (Du and Halkier, 1998). In Brassica, the levels of benzyl and hydroxybenzyl glucosinolates are often inversely correlated with each other, suggesting variation in the extent of hydroxylation and making it more likely that hydroxybenzyl glucosinolate is derived from phenylalanine (unpublished). In addition to hydroxy groups, one, two or three methoxy groups can also be added to the benzyl ring. Hydroxy groups can also be added to the aliphatic chain of phenylethyl glucosinolate, and are abundant in Barbarea species. Both R and S stereoisomers have been described (Huang et al., 1994). Indolyl glucosinolates derived from tryptophan can be modified through the addition of both hydroxy and methoxy groups. Uniquely, a sulphate group can be added (1-sulphonate-3-indolylmethyl glucosinolate). The abundance of this glucosinolate is difficult to monitor as the desulphonation of glucosinolates prior to HPLC analysis prevents this glucosinolate being distinguished from indolylmethyl glucosinolate. There is no apparent natural variation for indolyl glucosinolate modification inA. thaliana. Some variation occurs in Brassica but is yet to be explored. Thus, while several parts of glucosinolate biosynthesis remain to be resolved, significant progress has been made in recent years through molecular genetic studies inA. thaliana. With the full sequence of its genome now being available, and the provision of gene knockout mutants, the entire pathway is likely soon to be understood in considerable detail. E.

SITES OF B I O S Y N T H E S I S

At a cellular level, the location of glucosinolate biosynthesis has not yet been elucidated. However, some clues exist from cloned genes from A. thaliana. The MAM synthases, responsible for amino-acid elongation, have plastid targeting sequences, suggesting that elongation occurs in plastids. The CYP79 gene family possess sequences suggesting that they are targeted to the endoplasmic reticulum. At a tissue level, it is known that there is variation in glucosinolate content between leaves, roots and reproductive tissues, which may be due to different enzyme expression. Methioninederived glucosinolates in Brassica and hydroxybenzyl glucosinolate in Sinapis are not synthesized in the developing embryo, but in the silique wall and translocated into the seed during development (Kondra and Stefansson, 1970; Joseffson, 1971, 1973; Magrath and Mithen, 1993).

DIP

a-TIP

Merged

Plate 1. Association between dark intrinsic protein (DIP) organelles and PSV compartments in developing and mature tobacco seeds. Paraffin-embedded sections prepared from developing seeds at 10 days after pollination and mature seeds were doubled-labelled with anti-DIP (red, arrows) and anti-c~-tonoplast intrinsic protein (TIP) (green, arrowheads) antibodies and visualized by confocal immunofluorescence. In developing seeds, most DIP-labelled structures (red, arrow) were separated from developing PSVs (green, arrowhead), while in mature seeds, most DIP-labelled structures were contained within PSVs (green, arrowhead), u, nucleus; scale bar: 10 ~m.

a-TIP

V-PPase

Merged

E o

8

"o

,,=, (A)

(B) E

W

(c)

Plate 2. Labelling patterns for vacuolar pyrophosphatase (V-PPase) in different cell types within a tomato seed. Paraffin-embedded sections from a mature tomato seed were labelled with anti-~-TIP (green) and anti-V-PPase (red) antibodies and visualized by confocal immunofluorescence. Co-localization of the two antibodies in the merged images (right) is indicated by a yellow colour. (A) The labelling pattern for an endosperm cell. (B) The labelling pattern for cells comprising the outer layer of cortex in the radicle. Solid arrows, position of globoid cavities; open arrows, labelling for V-PPase along the PSV tonoplast. (C) The labelling pattern for cells immediately interior to the cells shown in (B). Solid arrows, position of globoid cavities. Scale bar: 10 ~m.

Plate 3. Image of a cross-section ofA. thaliana stem with superimposed X-ray maps of elements in false colours: S (red), K (green) and Ca (blue). Reproduced from Koroleva et al. (2000) Plant Physiology 124, 599-608, with permission from the American Society of Plant Physiology. Analytical studies have confirmed that the 'S' cells contain high levels of glucosinolates.

GLUCOSINOLATESAND THEIR DEGRADATIONPRODUCTS IV.

233

G E N E T I C A N A L Y S I S IN B R A S S I C A

A. MENDELIANGENESREGULATINGCHAINSTRUCTUREIN BRASSICA The diversity of glucosinolates within our major cruciferous crop species is relatively low compared to all possible structures that have been described. The greatest diversity is seen within B. oleracea (genome CC), especially when the wild species of the B. oleracea (n = 9) complex are included. Methinioine-derived glucosinolates in this species are restricted to either 3C or 4C side-chain lengths, but may comprise methylthioalkyl, methylsulphinylalkyl, alkenyl and hydroxyalkenyl side chains. Genetic analysis has enable several Mendelian genes, which determine side-chain structure to be positioned on linkage maps of B. oleracea and B. napus (Magrath et al., 1994; Parkin et al., 1994; Giamoustaris and Mithen, 1996). Thus alleles at Mendelian loci on linkage groups 2 and 5 have been shown to regulate chain length, alleles at loci on linkage group 9 regulate the oxidation of methylthioalkyl to methylsulphinylalkyl glucosinolates and the conversion of methylsulphinylalkyl to alkenyl glucosinolates, and alleles at loci on linkage group 3 regulate the hydroxylation of 3-butenyl glucosinolate. In contrast to the diversity observed in B. oleracea, B. nigra (genome BB) and the amphidiploid B. carinata (BBCC) have only 2-propenyl glucosinolate, and B. juncea (BBAA) either only 2-propenyl glucosinolate or a mixture of 2-propenyl and 3-butenyl glucosinolate. B. rapa (AA) and B. napus (AACC) lack 3C glucosinolates, and predominately possesses a mixture of 3-butenyl and 4-pentenyl glucosinolates and their 3-hydroxylated homologues, with much smaller amounts of methylsulphinylalkyl glucosinolates. Thus, all of these species can be considered to have functional alleles at the G S L - A L K locus, while some variation exists at E L O N G loci, which regulate the length of the side chain. Artificial forms of the amphidiploid B. napus can be made through the interspecific hybridization of B. rapa and B. oleracea. Thus, by selection of the appropriate parental glucosinolate genotype, the types of glucosinolates in B. napus can be altered. This has enabled the introduction of 2-propenyl GSL, the removal of hydroxyalkenyl glucosinolates and the reduction in the extent of 4-pentenyl glucosinolate (Giamoustaris and Mithen, 1995). Genetic analysis have position loci responsible for these alterations on to linkage maps, which have largely confirmed the position of genes mapped on homologous linkage groups in B. oleracea (Magrath et al., 1994; Parkin et al., 1994). Comparative mapping with cloned A. thaliana genes has suggested that the Brassica Mendelian genes are homologues of the cloned A. thaliana genes. Thus, DNA probes based upon the coding sequence of the MAM synthase genes cloned from A. thaliana identify RFLPs, which cosegregate

234

R.F. MITHEN

with E L O N G loci in Brassica regulating 3C, 4C and 5C glucosinolates. Thus the variation in chain length can be interpreted as allelic variation at several MAM synthase loci, similar to that for A. thaliana (Fig. 7). Likewise, probes based uponA, thaliana deoxygenase genes cosegregate with t h e A L K Brassica locus (Hall et al., 2000). B.

THE DEVELOPMENT OF LOW-GLUCOSINOLATE OILSEED RAPE: THE BRONOWSKI BLOCK AND QTL MAPPING

The last few decades has seen the emergence of Canola as a major oilseed crop throughout the temperate world. Its development required the reduction of two antinutritional factors in the seeds: erucic acid and glucosinolates. The elimination of erucic acid enabled the oil to be used for human consumption and the reduction in glucosinolates in the seed meal following oil extraction resulted in a high-quality animal feed. The need to reduce glucosinolates is due to the adverse effects of certain glucosinolate hydrolysis products on the feeding quality of the rapeseed meal, which remains following oil extraction from seeds. Of these products, 5-vinyloxazolidine-2-thione is of the greatest importance owing to its goitrogenic activity (see below for details). This compound is derived from 2-hydroxy-3butenyl glucosinolate, which accumulates in the seeds of oilseed rape. The low glucosinolate trait was identified in the Polish cultivar Bronowski (Josefsson and Appelqvist, 1968; Downey et al., 1969) and transferred into initially Canadian spring rape lines, and subsequently into European winter rape. Thus, glucosinolate levels have been reduced from above 80 ~mol g-1 to less that 5 ~mol g-1 in spring rape, and less than 15 ~mol g-1 in winter rape. While the major factor in this reduction is the genes introgressed from cv. Bronowski, undoubtedly genes from other cultivars have also contributed to the low levels of glucosinolates found in modern cultivars. The reduction in glucosinolates in Bronowski was restricted to methionine-derived glucosinolates. Josefsson (1971, 1973) undertook several biochemical studies to identify the nature of the metabolic block in glucosinolate biosynthesis in Bronowski through comparisons with highglucosinolate cultivars. It was showed that the low-glucosinolate character was not due to a reduction in sulphate uptake, or an increase in glucosinolate catabolism. Bronowski had an increase in the sulphur content of the silique wall (the site of synthesis of seed glucosinolates, see below). Feeding experiments with glucosinolate intermediaries suggested two metabolic blocks, firstly, between the synthesis of dihomomethionine and the oxime, and secondly, in the extent of hydroxylation of 3-butenyl glucosinolate. These biochemical studies were complemented by genetic studies, which indicated that the inheritance of glucosinolates was complex, and that several genes were involved (Kondra and Stefansson, 1970). These early studies were severely hampered by the analytical methods available, which

GLUCOSINOLATES AND THEIR D E G R A D A T I O N PRODUCTS

235

restricted the number of progeny that could be examined, and the interpretation of the data as some glucosinolates were not measured. Two developments have enabled the genetic basis of the "Bronowski block" to be further explored. Firstly, the development of recombinant double haploid lines of B. napus and linkage maps based upon restriction fragment length polymorphism (RFLP) molecular markers (e.g. Ferriera et al., 1994) enabled the genetic dissection of complex traits, and, secondly, advances in the analysis of glucosinolates, notably HPLC methods for the analysis of individual glucosinolates (Heaney and Fenwick, 1993), were of considerable help. Toroser et aL (1995) analysed quantitative trait loci (QTLs) in a double haploid population derived from a cross between a Stella, a low-glucosinolate cultivar, which had introgressed segments of the Bronowski genome, and Major, a high-glucosinolate winter oilseed rape cultivar. Two major QTLs were found that accounted for 33% and 21% of the variation in seed glucosinolates, and three minor QTLs, which accounted for a further 21% of the variation. No differences in the extent of hydroxylation were found between the two cultivars. However, there were differences in the ratio of 4C to 5C glucosinolates. QTLs that regulated this trait were coincident with QTLs that regulated total glucosinolates, suggesting a possible association. A further study by Uzunova et al. (1995) obtained similar results, locating four QTLs for glucosinolate content. The two most important QTLs were the same as those identified by Toroser et al. (1995). Comparative mapping studies with cloned genes from A. thaliana and their Brassica homologues should provide further information as to the nature of these QTLs. C.

ENHANCING GLUCOSINOLATES IN BROCCOLI

In contrast to glucosinolates that accumulate in rapeseed, the methylsulphinylalkyl glucosinolates, which accumulate in the florets of broccoli, are thought to be beneficial to health owing to the putative anticarcinogenic activity of their isothiocyanates (see below for details). Thus, there is now interest in enhancing these glucosinolates. Surveys of glucosinolates within exisiting broccoli cultivars have found variation of 4methylsulphinylbutyl glucosinolates from 0.8 ~mol g-~ to 21.7 ~mol g-~ dry weight (Kushad et al., 1999). However, in an effort to obtain higher levels, Faulkner et al. (1998) described the use ofB. villosa and other wild members of the B. oleracea (n = 9) species complex. Hybrids between these accessions and broccoli had levels in excess of 80 ~mol g-Z, but at the expense of agronomic characters. As with oilseed rape, the availability of linkage maps (e.g. Sebastian et al., 2000) is enabling QTLs, which regulate glucosinolate accumulation to be elucidated, and to be compared with those in B. napus.

236

R.F. MITHEN D. GENETICMODIFICATIONOF GLUCOSINOLATES

Genetic modification provides an additional route to altering both the levels and types of glucosinolates. As described above, the identification of glucosinolate biosynthesis genes from A. thaliana has involved phenotypic analysis following alterations of gene function. For example, constitutive expression of the CYP79A2 gene results in the synthesis of benzyl glucosinolate. Modification in the expression of MAM synthases has also led to changes in methionine-derived glucosinolates (G. Cardon, unpublished). Whether it will be possible to do similar modifications in the more complex Brassica genome, in which there are multiple copies of biosynthetic genes, remains to be seen. Other approaches may also be useful. For example, expression of a mammalian metallothionen in B. rapa led to a reduction in the level of glucosinolates (Lefebvre, 1990), and Chavedj et al. (1994) reports that indolyl glucosinolates can be reduced by redirecting tryptophan into tryptomine synthesis via the expression of tryptophan decarboxylase. V.

ENVIRONMENTAL FACTORS AFFECTING GLUCOSINOLATE EXPRESSION

In general terms, while the ratios of individual glucosinolates within a particular class (e.g. those derived from methionine) is relatively constant and unaffected by environmental factors, the total level is influenced by several environmental factors. Thus, while the total methionine-derived glucosinolate content of, for example, A. thaliana or Brassica leaves may often exhibit a 30-fold variation, the ratio of the individual glucosinolates, determined by the Mendelian genes described above, remains remarkably constant. As would be expected, nitrogen and sulphur supply affects the amounts of glucosinolates. Zhao et al. (1994) showed that sulphur and nitrogen supply affected glucosinolate content of rapeseed, and described minor alterations in the ratios of methionine-derived glucosinolates, and larger ones in the ratio of indolyl to methionine-derived glucosinolates. It has been suggested that glucosinolates can act as a sulphur store, so that in times of sulphur deficiency, sulphur and be mobilized from glucosinolates into primary metabolism (Schnug, 1989), although good evidence is lacking. The induction of glucosinolates following abiotic or biotic stresses has frequently been described. For example, pathogens (Doughty et al., 1995a), insect herbviores (Griffiths et al., 1994), salicylic acid (Kiddie et al., 1994), jasmonates (Bodnaryk and Rymeson, 1994; Doughty et al., 1995b) all induce glucosinolates. In general, indolyl glucosinolates seem to be induced to a greater extent and for a longer time compared with methionine-derived glucosinolates. The majority of these experiments have been undertaken on

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

237

glasshouse-grown plants in which glucosinolate expression is usually considerably less than plants grown in field experiments. Thus the ecological importance of this phenomenon is difficult to ascertain. Attempts to induce glucosinolates in field-grown oilseed rape plants by mechanical damage have not been successful (R. F. Mithen, unpublished). VI.

GLUCOSINOLATE DEGRADATION A.

MYROSINASES

Glucosinolates are one part of a two-component pre-formed defence system, which is activated upon tissue damage. Myrosinase, the enzymatic second component, is probably physically separated from the glucosinolates. When tissue is damaged, myrosinase comes into contact with glucosinolates, resulting in the cleavage of the thio-glucose bond and the generation of an array of products, which are described below. Bones and Rossiter (1996) and Rask et al. (2000) provide reviews of myrosinases, and only a brief summary is provided here. Many myrosinase isozymes have been detected in glucosinolatecontaining plants, and myrosinase (i.e. thioglucosidase) activity also detected in insects, fungi and bacteria. In plants, the expression of different isozymes varies both between species and between organs of the same individual (Lenman et al., 1993). As yet no correlation with activity or substrate specifity towards glucosinolate chain structure has been described. Molecular studies in A. thaliana, Brassica and Sinapis have shown that myrosinases comprise a gene family (Xue et al., 1992) within which there are three subclasses, denoted MA, MB and MC. Members of each of these subfamilies occur in A. thaliana (Xue et al., 1995). As would be expected, many more copies occur in the B. napus genome due to genome replication. All myrosinases are glycosylated, and the extent of glycosylation varies between the subclasses. It is likely that the subdivision of myrosinases will be revised as new sequence data become available. Associated with MB and MC myrosinases are myrosinase-binding proteins (MBPs; Lenman et al., 1990). This is a large class of proteins with masses ranging from 30 to 110 kDa, and often contains sequences of aminoacid repeats. At least 17 MBPs have so far been found inA. thaliana. Some of the MBPs have lectin-like activity (Taipalensuu et al., 1997). The role of these proteins is far from understood. They are induced upon wounding or by application of jasmonates (Geshi and Brandt, 1998). It has been suggested that they may stabilize the myrosinase enzymes themselves, or possibly interact and bind to carbohydrates on the surface of invading pathogens. In addition, another class of glycoproteins, designated myrosinase-associated proteins (MyAP) has also been identified in

238

R.F. MITHEN

complexes with myrosinase and MBPs. As with MBPs, the role of these is not understood. They have sequence similarities to lipases. Isoforms that occur in leaves are inducible upon wounding or treatment with methyl jasmonate (Taipalensuu et al., 1996). B. CELLULARAND SUBCELLULARLOCATIONOF GLUCOSINOLATESAND MYROSINASES Despite many studies, and considerable speculation, the precise organization of the glucosinolate-myrosinase system is not fully understood. Myrosinases occur in specialized cells ("myrosin" cells), which occur in most tissues at a frequency of between 2% and 5% (Bones and Iverson, 1985). Within these cells, protein-rich granules are found (myrosin grains), which were presumed to be the location of the myrosinase enzyme. Immunohistochemical studies have confirmed the presence of myrosinases in these specialized cells (Thangstad et al., 1990, 1991; H6gland et al., 1991), and have shown the enzyme to be located to the interior of the cell. However, tissues that lack myrosin cells still have myrosinase activity and thus it is likely that myrosinases may additionally occur in the cytoplasm of nonspecialized cells (Thangstad et al., 1991). The location of glucosinolates is even less well understood. It is likely that they reside within the cell vacuole (Grob and Matile, 1979). The glucose and sulphate moieties may aid the transportation of glucosinolates into the cell vacuoles, probably via an active transport mechanisms. In seeds, 2-propenyl glucosinolate has been associated with protein bodies in non-myrosin cells (Kelly et al., 1998). However, the extent to which glucosinolates are concentrated in certain cell types in vegetative tissues is not known. Koroleva et al. (2000) have described a specialized cell type ("S cells") in the stems ofA. thaliana situated between the phloem and the epidermis, which are very rich in sulphur and contain high concentrations of glucosinolates (Plate 3). Liithy and Matile (1984) has termed this two-component system the "mustard oil bomb", in which myrosinase occurs in the cytoplasm and glucosinolates in the vacuole of the same cells, awaiting detonation by cellular damage. Even if this model needs to be revised with the glucosinolates being in specialized cells, the analogy is still appropriate. C. PRODUCTS When tissue disruption occurs, myrosinase activity results in the cleavage of the thio-glucose bond of the glucosinolate to give rise to unstable thiohydroximate-O-sulphonate. This aglycone spontaneously rearranges to produce several products (Fig. 9a). Most frequently, it undergoes a Lossen rearrangement to produce an isothiocyanate. If the isothiocyanate contains

239

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS

~

(a)

-

S--Glucose

II

N"-oso#

/SH

~

R~NzC~S

Isothiocyanate

}, R - - C ~ N

N"'OSO3-

" ~

Nitrile

~--S--C~N Thiocyanate

unstable intermediate

Glucosinolate

(b) S--Glucose C H ~ CH--- CH2----CH2~C/

II

3-butenyl glucosinolate

N...OSO3_

C H ~ CH--CH2----CH2~ N~---C ~ S 3-butenyl isothiocyanate

~ Epithiospecifierprotoin CH2~CH--CH2~CH~---C~ N

\/

(C)

OH

S--Glucose

I

CH2~ CH~CH--CH2-~C / 2-hydroxy-3-butenyl glucosinolate

CH2~---CI-I~CH

I[

N"'OSO3-

?H 2

I

O~cT/NH II

S

5-vinyloxazolidine-2-thione

S 1-cyano-3,4-epithiobutane

Fig. 9. (a) Degradation of glucosinolates. (b) Conversion of an isothiocyanate to an epithionitrile due to presence of the epithiospecifier protein. (c) Conversion of 2hydroxy-3-butenyl glucosinolate to the goitrogenic compound 5-vinyloxazolidine-2thione.

a double bond, and in the presence of an epithiospecifier protein (see below), the isothiocyanate may rearrange to produce an epithionitrile (Fig. 9b). At lower pH, the unstable intermediate may be converted directly to a nitrile with the loss of sulphur. Conversion to nitriles is also enhanced in the presence of ferrous ions (Uda et aL, 1986). A small number of glucosinolates have been shown to produce thiocyanates, although the mechanism by which this occurs is not known. Aglucones from glucosinolates, which contain ,6hydroxylated side chains, such 2-hydroxy-3-butenyl ("progoitrin") found in the seeds of oilseed rape, spontaneously cyclize to form the corresponding oxazolidine-2-thiones (Fig. 9c). Indolyl glucosinolates also form unstable isothiocyanates, which degrade to the corresponding alcohol and may condense to form diindolylmethane. At more acidic pH, indolyl

240

R.F. MITHEN

glucosinolates can form indolyl-3-acetonitrile and elemental sulphur (Fig. 10). This nitrile has auxin activity and can also be converted to indole-3acetic acid. The epithiospecifier protein (ESP) was first described by Tookey (1973), and purified from B. napus (Bernardi et al., 2000; Foo et al., 2000). This protein appears to have no enzymatic activity of its own, and does not interact with glucosinolates, but only the unstable thiohydroximate-Osulphonate following myrosinase activity. Foo et aI. (2000) suggest that ESP has a mode of action similar to a cytochrome P450, such as in iron-dependent epoxidation reactions. While this protein has only been considered in the context of the generation of epithionitriles, it may be involved in the production of nitriles from glucosinolates such as methylsulphinylalkyls. In this case, the sulphur from the glucone is lost as it can not be re-incorporated into the degradation product owing to the lack of a terminal double bond. There has been little quantitative work on glucosinolate degradation, mainly due to the difficulties of quantifying volatile isothiocyanates. Semi-

_S--Glucose

"oso I

R Indolylglucosinolate

/ ~ ~ 1 ~

½

cH2-OH +

SCN

I

R

Indolyl-3-carbinol

Indolyl-3-acetonitrile

C H 2 ' ~ ~

I

~

/CH2--C~ N

]

R

R

R = H or OCH3

I

R

Diindolylmethane

Fig. 10. Degradation ofindolyl glucosinolates.

241

GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS IsothJocyanate (ITC)

R--- N ~ C ~ S Glutathione transferases or non-enzymic

ITC-glutathione conjugate

0 0 II II R - - N H - - C,~ S - - CH2-~ C H - - C - - N H - - C H2---C,~OH

II

S

I

N H - - C,~ C H 2 ~ C H 2 ~ C H---C,--- OH

II

~,

ITC-cysteineglycine conjugate

O

II

0 0 II II R--- N H-- C---S-- CH2--- CH-- C---NH--CH2--- C---OH

II

S

ITC-cysteine conjugate

I

NH 2 0

I

NH2

O II R--- NH--C---S--CH2--CH-- O - - O H

II

S

I

NH 2

0

II

ffC-N-acetylcysteine conjugate

R - - N H - - C - - S - - C H2-- CH--- C - - OH

II

S

;

I

NH--C---CH3

oII

Excretion

Fig. 11. Human metabolism of isothiocyanates. Analysis of N-acetylcysteine conjugates in urine provides a useful marker for isothiocyanate metabolism.

quantitative studies have suggested a very large range in the amount of isothiocyanates and other products being generated by a unit amount of glucosinolates, and that this variation appears to have a genetic basis and is not simply related to the myrosinase activity. Likewise, in addition to variation in hydrolysis conditions (e.g. pH, ferrous ions), there is distinct interspecific as well as intraspecific variation in the relative production of isothiocyanates and nitriles, which may be related to the expression of ESP. D.

METABOLISM AND DETOXIFICATION OF ISOTHIOCYANATES

Isothiocyanates are biologically active compounds. The sulphur of the isothiocyanate group enables the formation of disulphide links with proteins

242

4. F. MITHEN

and other metabolites, which can have major deleterious effects. Mammals metabolize isothiocyanates by initial conjugation with glutathione, and then subsequent metabolism via the mercapturic acid pathway, and excretion as N-acetylcysteine-isothiocyanate conjugates (Fig. 11; Brusewitz et al., 1977; Mennicke et al., 1983, 1988). The presence of N-acetylcysteineisothiocyanate conjugates in urine provides a useful marker for isothiocyanate metabolism (Duncan et al., 1997; Chung et al., 1998; Sharpio et al., 1998; Rose et al., 2000). Epithionitriles are also metabolized in a similar manner (Brocker et al., 1984). The metabolism of other glucosinolate degradation products is not known. Nitrile metabolism if of particular interest owing to the potential to generate antinutritional compounds via cytochrome P450-mediated metabolism Isothiocyanates generated within plant tissue also need to be detoxified. This may be by conjugation with glutathione, and then transportation into the vacuole where they may be further metabolized. Glutathioneisothiocyanate conjugates can be found in crude plant extracts (Rose et al., 2000), but their biological importance is not clear. Likewise, pathogenic fungi and insect herbivores may also detoxify isothiocyanate by glutathione conjugation, although there is no experimental evidence for this at present. VII.

BIOLOGICAL ACTIVITY OF GLUCOSINOLATES AND THEIR DEGRADATION PRODUCTS A.

PLANT-ANIMAL INTERACTIONS

The evolutionary origins of glucosinolates is likely to be related to their presumed role in plant defence against herbivores. While it is likely that the evolution of glucosinolates is connected to cyanogenic glycoside synthesis, there is very little understanding concerning how and when glucosinolates first appeared. To provide any deterrent, the thioglucosidase must also be present. Thus, it is likely that earlyBrassicale-like plants may have made small amounts of glucosinolates, which were hydrolysed following evolutionary changes in existing glucosidases to produce isothiocyanates, which acted as a novel defence, providing an increase in fitness over non-glucosinolateisothiocyanate-producing plants. Subsequent chain diversification through the recruitment of novel amino-acid precursors and chain modifications may have been driven by an "arms race" between plants and insects, which become adapted to existing glucosinolate chain structures (see below). While this is an attractive hypothesis, there is little evidence for it, and chain diversity may have evolved through random events and genetic drift, conferring no selective advantage. Despite the considerable amount of speculation, there is relatively little evidence for the role of glucosinolates in effective defence against

GLUCOSINOLATESAND THEIR DEGRADATIONPRODUCTS

243

herbivores and pathogens. Numerous experiments have documented induction of glucosinolates (usually indolyl glucosinolate) following damage or by elicitation with jasmonates, and assumed that these are important defensive compounds. The majority of studies have concentrated on the manner by which isothiocyanates act as attractants, and feeding and egglaying stimulants for "specialized" insects, and in vitro studies of the toxicity of isothocyanates towards fungal and bacterial pathogens. The main defensive role of glucosinolates is likely to be in the manner by which they deter generalist herbivores such as molluscs, and probably has its greatest effectiveness at the time of seedling recruitment. Giamoustaris and Mithen (1995) demonstrated that increasing the levels of glucosinolates in leaves of Brassica resulted in less damage by molluscs and birds (but enhanced damage by insect herbivores; see below). Likewise, lines of Brassica that had effectively lost aliphatic glucosinolates were heavily and selectively grazed by an assortment of birds and small mammals (Mithen, 1992). Glen et al. (1990) demonstrated that slugs preferentially grazed seedlings with low levels of glucosinolates. Blau et al. (1978) showed that the growth of two generalist insect herbivores were inhibited by increasing glucosinolate concentration, in contrast to the specialist Pieris rapae. In a similar manner, the Bertha army worm, Mamestra configurata, fed more on leaves with low glucosinolates than those with higher amounts (Bodnaryk, 1997). Thus, continual reduction of glucosinolates in the leaves of Canadian spring rape may result in increased pest damage by insects that are not usually problematic for crucifers. The majority of studies have been on the manner by which glucosinolates and isothiocyanates attract and stimulate feeding and egg laying of insects, which are specialized feeders of the Brassicaceae. These studies have adopted two main approaches. Firstly, several studies have shown positive correlations between glucosinolate content and insect damage (Giamoustaris and Mithen, 1995; Siemens and Mitchell-Olds, 1996; Lambdon et al., 1999), although herbivory may be reduced at high concentrations. Secondly, experimental studies have investigated behavioural and neurological responses of insects to glucosinolates and isothiocyanates. Many insects have been shown to be stimulated and attracted to volatile isothiocyanates, e.g. Psylliodes spp. (Blight et al., 1989; Pivnick et al., 1992; Isodoro et al., 1998), Ceutorhynchus spp. (Bartlet et al., 1993), Dasineura brassicae (Murchie et al., 1997), Plutella xylosa (Pivnick et al., 1994). This has led to the use of isothiocyanates as bait in traps (e.g. Burgess and Wiens, 1980). Likewise, insects also use isothiocyanates as feeding and ovipositioning stimulants, e.g. Pieris spp (Huang et al., 1994; Stadler et al., 1995), Psylliodes chrysocephala (Bartlet et al., 1994) and Delia floralis (Simmonds et al., 1994; Braven et al., 1996; Roessingh et al., 1997). In many of these cases, other plant compounds may also be important and may act synergistically with glucosinolate derivatives.

244

a.F. MITHEN

Alterations in chain structure have important effects upon these interactions. Firstly, minor chemical alterations can have major effects on the physiochemical properties of the products. For example, while 4-methylthiobutyl glucosinolate degrades to a volatile isothiocyanate, 4methylsulphinylbutyl glucosinolate produces a non-volatile isothiocyanate, with consequences for long-range insect signalling. Likewise, while 3butenyl glucosinolate produces a volatile isothiocyanate, 2-hydroxy-3butenyl glucosinolate is hydrolysed to a non-volatile thione. Insects have shown adaptation to specific glucosinolates. For example, insects pest of oilseed rape are more responsive to 3-butenyl and 4-pentenyl isothiocyanate, which will be generated from leaves of oilseed rape, than to 2-propenyl isothiocyanate, which is not produced by B. napus. Subtle changes can also be important, for example, Pieris species are able to differentiate between R and S epimers of hydroxy-phenylethylglucosinolate (Huang et al., 1994). Parasites of insect herbivores are also attracted by isothiocyanates (Reed et al., 1985; Vaughn et al., 1996; Murchie et al., 1997). For example, Diaeretiella rapae, is attracted by isothiocyanates, but in particular 3-butenyl isothiocyanate. Increasing the production of this isothiocyanate in B. napus led to more aphid parasitism in field experiments (Bradburne and Mithen, 2000). Thus, increasing glucosinolate content may lead to protection from generalist herbivores while increasing susceptibility to specialist herbivores, in addition to the unknown metabolic cost involved in synthesizing and storing glucosinolates and myrosinase. Induction of glucosinolates by biotic and abiotic factors may also be of importance in mediating these interactions (Bodnaryk and Rymerson, 1994; Bartlet et al., 1999). There is some evidence that, in wild plant populations glucosinolate content is driven by selection from herbivores. Population genetic studies suggested that the contrasting glucosinolate content of plants of two adjacent populations of wild Brassica oleracea was likely to be due to differential herbivory (Mithen et al., 1995b). Ecological studies suggested that variation in glucosinolate content between populations was unlikely to be due to differential herbivory on mature plants (Moyes et al., 2000). It is possible that it is during the seedling recruitment stage that differential herbivory due to glucosinolate variation has important effects on plant survival and fitness. Correlations between insect damage, soil moisture content and the methylglucosinolate content of the Cleome serrulata have been reported (Louda et al., 1987), illustrating complex interaction between environmental factors, secondary metabolism and plant defence. While the majority of glucosinolate-containing plants in temperate ecosystems are ruderals or short-lived perennials, in tropical and sub tropical ecosystems many glucosinolate-containing plants are shrubs and small trees. The defensive ecology of these plants has been largely unexplored. Many

GLUCOSINOLATESAND THEIR DEGRADATIONPRODUCTS

245

glucosinolate-containing genera of the Capparaceae, such as Maerua, Boscia and Capparis occur in heavily grazed parts of sub-Saharan Africa (R. F. Mithen, personal observations). The metabolic costs of the glucosinolate-myrosinase system has been considered by Mauricio and Rauscher (1997), who described selection in A. thaliana for higher glucosinolates plants in plants exposed to herbivory, although at a cost to seed reproduction. Likewise, Siemens and Mitchell-Olds (1998) suggested that plants with a high level of constitutive expression of myrosinase were associated with a decrease in seed production. An interesting and relatively unexplored factor is the maternal inheritance of glucosinolate and its implications for plant-herbivore interactions. As discussed above, methionine-derived glucosinolates in the seeds are maternally inherited. Moreover, they are not synthesized in the cotyledon, but only in subsequent true leaves. Hence, the crucifer seedling expresses glucosinolates synthesized in the siliques of the maternal plant. In addition to the maternal genotype (which will be different from that of the zygote and seedling), environmental factors that may have altered the glucosinolate content of maternal siliques, such as induction due to damage or insect attack, or availability of nitrogen and sulphur, will have consequences for herbivore interactions with seedlings, at which stage plants are probably most vulnerable. This maternal inheritance of glucosinolates underlies the observations of Agrawal et al. (1999). The introduction of low-glucosinolate oilseed rape led to concern that it may be more susceptible to herbivore damage than its high-glucosinolate precursor (Mithen, 1992; Bodnaryk, 1997). In Europe, the early lowglucosinolate winter oilseed rape cultivars were highly palatable to herbivores such as deer, pigeons and rabbits, while subsequent cultivars were similar to high-glucosinolate cultivars. Mithen (1992) showed that these early cultivars had lost glucosinolates throughout their vegetative and reproductive tissues, while the latter cultivars retained glucosinolates in overwintering vegetative tissues, but synthesis appeared to cease at about the onset of flowering. B. PLANT-PATHOGENINTERACTIONS Glucosinolates are frequently quoted in the scientific literature as affording disease resistance. There is no evidence for this. In vitro studies have shown that isothiocyanates and other glucosinolate degradation products are toxic to fungi and bacteria, including pathogens ofBrassica (Drobnica et al., 1967; Mithen et al., 1986; Sarwar et al., 1998). However, enhancing glucosinolate content in leaves of Brassica does not confer increased resistance to Brassica pathogens, and possibly results in greater susceptibility (Giamoustaris and

246

R.F. MITHEN

Mithen, 1997). These pathogens may have evolved to be able to tolerate and detoxify glucosinolates, in a similar manner to insect herbivores. Thus, the response of pathogens to glucosinolates and their products in planta may depend on whether the pathogen is a specialized Brassica pathogen, such as Leptosphaeria maculans, or a more generalist pathogen, such as Botrytis cinerea. As with plant-insect interactions, several pathogens, including Plasrnodiophora brassicae, the cause of clubroot disease, have been shown to induce indolyl glucosinolates (Butcher et al., 1974). This has often been referred to as a "defence" response. However, there is no evidence to suggest the involvement of these compounds in reducing the growth of the invading pathogen. Many pathogens of crucifers cause tissue deformities, the most obvious ones being clubroot andAlbugo brassicae. This is likely to be due to induction of auxin biosynthesis, which may itself be related to induction of indolyl glucosinolates as both pathways share intermediates (see above). However, indolyl glucosinolates can themselves have auxin-like activity (Skytt Andersen and Muir, 1966). Crucifers have a long history as green manure crops. This practice adds organic matter and minerals to soils, but probably also has a positive effect on pests and pathogens for future crops by reducing the inoculum potential, a phenomenon currently referred to as "biofumigation". Cereal crops often show an increase in yield of up to 10% when following a oilseed rape or mustard crop (Kirkegaard et al., 1994). This has often been thought to be due to residual nitrogen, but it is likely that it is at least due in part to the effect of isothiocyanates released from growing roots of the oilseed crop on the microflora of the soil. Isothiocyanates are toxic to many soil-borne pathogens of a variety of crops (Sarwar et al., 1998), and when released from roots in laboratory experiments can inhibit growth of important cereal pathogens such as take all (Angus et al., 1994). Gardiner et al. (1999) identified several glucosinolate degradation products in the soil after ploughing in vegetative rapeseed crops. C. FLAVOUR The presence of glucosinolates in horticultural cruciferous crops is important both for flavour and as potential anticarcinogens. Their importance as flavour compounds is well known. Isothiocyanates provide the characteristic hot and pungent flavours of many of our cruciferous salad crops and condiments. Likewise isothiocyanates and other glucosinolate degradation products are important flavour components of cooked cruciferous vegetables. The contribution of these to flavour is, however, complex. Bitterness in some cultivars of Brussels sprouts is possibly due to levels of 2-hydroxy-3-butenyl and 3-butenyl glucosinolates, but the direct cause is difficult to elucidate.

GLUCOSINOLATESAND THEIR DEGRADATIONPRODUCTS

247

D. ANTINUTRITIONALEFFECTS IN LIVESTOCKAND HUMANS The presence of glucosinolates in the seeds of oilseed cruciferous crops significantly reduces the livestock feeding quality of the meal left following oil extraction from seeds (Bell, 1984; Griffiths etal., 1998). This is largely due to the presence of 2-hydroxy-3-butenyl glucosinolate, which degrades to 5vinyloxazolidine-2-thione, which causes thyroid disfunction by acting as an inhibitor of thyroxine synthesis (Elfving, 1980). With the expression of ESP, this glucosinolate can also degrade to 1-cyano-2-hydroxy-3-butene, which can result in enlargement of liver and kidneys (Gould et al., 1980). Thiocyanates, which can also be derived from glucosinolates, can also have goitrogenic activity by acting as iodine competitors. Plant myrosinases are not important for these antinutritional effects as gut bacteria has sufficient thioglucosidase activity to degrade glucosinolates (Oginsky et al., 1965). There is no evidence for any goitrogenic effect of Brassica consumption in humans; inclusion of 150 g of Brussels sprouts in the diet of adult volunteers had no effect on thyroid hormones (McMillan et al., 1986). As there is currently interest in increasing Brassica consumption due to putative anticarcinogenic activity, it is important to resolve the possibility of antinutritional effects of Brassica in humans. Some glucosinolate products have been implicated as possible carcinogens. Foremost amongst these are indolyl-3-carbinol and indole-3acetonitrile from hydrolysis of indolyl glucosinolates. These can be metabolized in the presence of nitrite to form mutagenic N-nitroso compounds (Wakabayashi et al., 1985; Tiedink et al., 1991). 2-Propenyl and phenylethyl isothiocyanates have also been shown to induce clastogenic changes in mammalian cell lines (Musk et al., 1995a,b). However, considerable amounts of epidemiological data and experimental studies suggest that glucosinolate and their hydrolytic products act as anticarcinogenic agents in the diet. E. ANTICARCINOGENICACTIVITY Diets rich in crucifers probably protect humans against a variety of cancers, particularly of the gastrointestinal tract (World Cancer Research Fund, 1997). This is likely to be largely due to the biological activity of glucosinolate degradation products. Two main modes of action have been investigated; modulation of phase I and phase II enzyme activity, and induction of apoptosis. Phase I and II enzymes metabolize xenobiotics and play a major role in preventing carcinogenesis. Phase ! enzymes, mainly cytochrome P450s, are usually considered to be activating enzymes, which initiate conversion of lipophilic xenobiotics into more hydrophilic metabolites, enabling them to be conjugated with a variety of metabolites (e.g. glutathione, sulphate,

248

a.F. MITHEN

glucuronic acid, etc.) prior to excretion via phase II enzyme activity (see below). Thus, induction can lead to the decrease of exposure of tissues to the parental xenobiotic. However, the resultant intermediate metabolites are often highly reactive, and can form DNA adducts, oxidative stress and be cytotoxic, with an associated risk of cancer. Thus, excessive phase I activity, without associated phase II activity, can be undesirable and may enhance cancer risk, while moderate phase I activity may be beneficial. Phase II enzymes, such as glutathione transferases, NAD(P)H:quinone reductase, UDP-glucuronylsyltransferases and sulphotranferases, catalyse conjugation reactions leading to the further metabolism and excretion of the xenobiotics prior to toxic effects. The induction-inhibition of phase I and induction of phase II enzymes is probably one of the main mechanisms that underlies the anticarcinogenic effects of fruit and vegetables in the diet (Block et al., 1992). Isothiocyanates, depending upon chain structure, can act as inhibitors of phase I and inducers of phase II enzymes, while degradation products of indolyl glucosinolates can induce phase I enzymes. Induction of phase II enzymes by isothiocyanates has been well documented, partly because of well-established cell-culture assays, which have been validated against animal models (Prochaska et al., 1992; Tawfiq et al., 1995). These measure the induction of quinone reductase in murine hepatoma cell cultures in 96-well microtitre plates. While several isothiocyanates have been shown to induce phase II enzymes, 4methylsulphinylbutyl isothiocyanate ("sulphoraphane") has been shown to be a particular potent inducer, both in cell culture assays and in animals (Zhang et al., 1992, 1994). Furthermore, administration of sulphoraphane to rats treated with the carcinogen 9,10-dimethyl-l,2-benzanthracene reduces the incidence and sizes of tumours. Other methylsulphinylalkyl isothiocyanates, such as 7-methylsulphinylheptyl and 8-methylsulphinyloctyl isothiocyanates found in watercress, are also potent inducers (Rose et al., 2000). Isothiocyanates of other chain structures can also induce phase II enzymes. For example, Wallig et al. (1998) reported that a mixture of glucosinolate breakdown products in Brussels sprouts induces glutathione transferases in rats, and Van Leishout et al. (1996) demonstrated induction of glutathione transferases in rats by phenylethyl isothiocyanate. Some isothiocyanates inhibit phase I enzymes (Guo et al., 1992, 1993; Conaway et al. 1996). For example, phenylethyl isothiocyanate is thought to inhibit the rodent lung carcinogen 4-(methylnitrosamino)-l-(3-pyridyl)-lbutanone (NKK) by blocking its activation (Schulze et al., 1995; Hecht et al., 1996; Chung et al., 1998). Despite the evidence for inhibition of phase I enzymes, crucifer consumption has been reported to cause an induction of the phase I enzyme CYP1A2, as determined by the analysis of caffeine metabolites in urine (Kall et al., 1996; Lambe et al., 2000). This is likely to be due to indolyl glucosinolate degradation products. For example, indole-3carbinol is a weak inducer of CYPIA1 in cell cultures, but is a more potent

GLUCOSINOLATESAND THEIR DEGRADATIONPRODUCTS

249

inducer in vivo after oral administration, owing to its conversion to indolo[3,2-b]carbazole and 3,3'-diinodolylmethane (Chen et al., 1996). In contrast, N-methoxyindole-3-carbinol, derived from N-methoxyindolyl glucosinolate, which is often more prevalent in Brassica vegetables than indolylmethyl glucosinolate, is a more efficient inducer in cell cultures of CYP1A1 than when administered to rats (Bradfield and Bjeldanes, 1987; Stephensen et al., 2000). The induction and inhibition of phase I and II enzymes by crucifers is also likely to depend upon the genotype of the consumer, particularly at the loci that code for glutathione-S-transferases, such as GSTM1 and the GSTT1 loci. Zhang et al. (1995) showed that GSTM1 is very potent at catalysing the conjugation of glutathione with isothiocyanates. Thus, it is possible that people who are GSTM1 null, i.e. approximately 40% of the population (Cotton et aL, 2000) will excrete isothiocyanates at a slower rate, maybe enabling greater enzyme inhibition-induction by isothiocyanates. This hypothesis is supported by the study of Probst-Hensch et al. (1998) who reported that cytochrome P450 activity amongst frequent consumers of crucifers was related to their GSTM1 genotype. The relationship between GSTM1 genotype and enzyme induction may underlie the finding that broccoli had a protective effect for the development of colorectal adenomas only for people with a GSTM1 null genotype (Lin et al., 1998). The complex interaction between incidence of colon cancer, GST genotype, crucifer consumption and other factors is further illustrated in the study of Slattery et al. (2000). Glucosinolate degradation products may also suppress tumour development after initiation of carcinogenesis, although the mechanisms by which this occurs are far from understood. For example, 2-propenyl isthiocyanate is selectively cytotoxic to cancer cells in vitro (Musk and Johnson, 1993; Musk et al., 1995). 2-Propenyl, 4-methylsulphinylbutyl and phenylethyl isothiocyanates can induce apoptosis in cancer cell cultures (Chen et al., 1998; Huang et al., 1998; Smith et al., 1998; Yu et al., 1998; Gamet-Payrastre et al., 2000; Xu and Thornalley, 2000). It is likely that isothiocyanates have other modes of activity to inhibit tumorigenesis (e.g. Morse et al., 1993). The majority of Brassica vegetables contain mixtures of glucosinolates, which may produce an array of products that are going to interact with each other and with other plant-derived metabolites. The beneficial effects of broccoli may be associated with it possessing a moderate amount of indolyl glucosinolates combined with 4-methylsulphinylbutyl glucosinolate; the indolyl glucosinolate degradation products enabling induction of phase I enzymes, which provide substrates from phase II activity induced by 4methylsulphinylbutyl isothiocyanate. The relationship between crucifer consumption and reduction in risk from cancer is complex. The extent of exposure of tissues to glucosinolate

250

R.F. MITHEN

degradation products are going to depend upon genetic and environmental factors determining the level of glucosinolates in the plant and their conversion to isothiocyanates, either by plant or gut microbial myrosinases, and the effect of processing (blanching, freezing, cooking, etc.). The responses of tissues are going to depend upon the genotype at GSTM1 (and probably other loci) and a host of interacting factors, which are unknown at present. These factors will then interact with risk factors such as exposure to environmental and dietary carcinogens. VIII.

CONCLUSIONS

Considerable advances have recently been made in our understanding of the biosynthesis and biology of glucosinolates and their degradation products. What will be the next major developments in glucosinolate research? There are probably three areas in which there will be significant progress over the next few years. Firstly, the complete biosynthetic pathway will be described, including the enzymatic conversion of the oxime to a thiohydroximate. This will most likely be achieved through the analysis of mutants in A. thaliana, and will proceed alongside the description of the genes and associated genetic modification. Secondly, there will be a greater understanding of the genes underlying quantitative trait loci controlling glucosinolate accumulation in complex genomes such as Brassica. These may be structural genes coding for enzymes of the biosynthetic pathway or they may be genetic elements such as transcription factors. Thirdly, there was be significant advances in understanding how isothiocyanates, and other glucosinolate products, mediate the metabolism of xenobiotics, apoptosis and other interactions with mammalian cells leading to reduced cancer risk. ACKNOWLEDGEMENTS I am grateful to the John Innes Centre, the Biotechnology and Biological Research Council and the University of Nottingham for financial support. I would like to express my thanks to Ruth Magrath and all other past and present members of my research group. REFERENCES Agrawal A. A., Laforsch, C. and Tollrian, R. (1999). Transgenerational induction of defences in animals and plants. Nature 401, 60-63. Angiosperm Phylogeny Group (1998). An ordinal classification for the families of flowering plants. Annals of the Missouri Botanical Garden 85, 531-553. Angus J, F., Gardner P. A., Kirkegaard J. A. and Desmarchelier J. M. (1994). Biofumigation, isothiocyanates release from Brassica roots inhibit growth of the take-all fungus. Plant and Soil 162, 107-112.

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