The biosynthesis of glucosinolates

reviews 26 Gillham, N.W. (1994) Organelle Genes and Genomes, Oxford University Press 27 Fischer, N. et al. (1996). Selectable marker recycling in the chloroplast, Mol. Gen. Genet. 251, 373-380 28 Malign, P. (1994) Isolation and characterization of mutants in plant cell culture, Annu. Rev. Plant Physiol. 35, 519-542 29 Harada, T. et al. (1992) Pollen-derived rice calli that have large deletions in plastid DNA do not require protein synthesis in plastids for growth, Mol. Gen. Genet. 233, 145-150 39 Gruissem, W. and Tonkyn, J.C. (1993) Control mechanisms of plastid gene expression, Crit. Rev. Plant Sci. 12, 19-55 31 Fong, S.E. and Surzycki, S.J. (1992) Chloroplast DNA polymerase genes of ChIamydomonas reinhardtii exhibit an unusual structure and arrangement, Curr. Genet. 21, 485-487 32 Winter, U. and Feierabend, J. (1990) Multiple coordinate controls contribute to a balanced expression of ribuiose 1,5-bisphospate carboxylase/oxygenasesubunits in rye leaves, Eur. J. Biochem. 187, 445-453 33 Huang, C. et al. (1994) The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth, Mel. Gen. Genet. 244, 15t-159 34 Allison, L.A., Simon, L.D. and Maliga, P. (1996) Deletion of rpoB reveals a second distinct transcription system in plastids of higher plants, EMBO J. 15, 2802-2809 35 Wolfe, K.H., Morden, C.W. and Palmer, J.D. (1992) Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant, Proc. Natl. Acad. Sci. U. S. A. 89, 10648-10652 36 Hess, W.R. et al. (1993) Chloroplast rps15 and the rpoB/CiC2 gene cluster are strongly transcribed in ribosome-deficientplastids: evidence for a functioning non-chloroplast-encodedRNA polymerase, EMBO J. 12, 563-571 37 Lerbs-Mache, S. (1993) The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single subunit enzyme or catalytic

core of multimeric enzyme complexes?Proc. Natl. Acad. Sci, U. S. A. 90, 5509-5513 38 Mullet, J.E. (1993) Dynamic regulation of chloroplast transcription, Plant Physiol. 103, 309-313 39 Shapiro, J.A. (1993) A role for the Clp protease in activating Mumediated DNA rearrangements, J. Bacteriol. 175, 2625-2631 49 Boudreau, E. et al. (1997) A large open reading frame (ORF 1995) in the chloroplast DNA of Chlamydomonas reinhardtii encodes an essential protein, Mol. Gen. Genet. 256, 649-653 41 Taylor, W.C. (1989) Regulatory interactions between nuclear and plastid genomes, Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 211-233 42 Takahashi, Y. et al. (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I, EMBO J. 10, 2033-2040 43 Kuras, R. and Wollman, F-A. (1994) The assembly of cytochromeb6/f complexes:an approach using genetic transformation of the green alga Chlamydomonas reinhardtii, EMBO J. 13, 1019-1027 44 Kanevski, I. and Maliga, P. (t994) Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-l,5 bisphosphate carboxylase in tobacco chloroplasts, Proc. Natl. Acad. Sci. U. S. A. 91, 1969-1973 45 Rochaix,J-D. (1995) Chlamydomonas reinhardtii as the photosynthetic yeast, Annu. Rev. Genet. 29, 209-230 46 Sugita, M. et al. Targeted deletion ofsprA from the tobacco plastid genome indicates that the encoded small RNA is not essential for pre16S rRNA maturation in plastids, Mol. Gen. Genet. (in press)

J6anlDavid Rocha:ixis a t ihe Depts

M~ie:~iai BioiogYaad

Geae#a;:Swi~etiand

The biosynthesis of glucosinolates Barbara Ann Ha kier and Liangcheng Du Glucosinolates and their hydrolytic products have a wide range of effects that are of biological and economic importance. In particular, these compounds have been shown to mediate interactions between plants and pests, and to reduce the feeding quality of rapeseed meal. Significant progress has now been made in understanding the biochemistry and genetics of glucosinolate biosynthesis, and the first Arabidopsis genes involved should soon be isolated. Hence, modifying the level of glucosinolates in Brassica crops, both to study interactions with pests and to improve flavour and nutritional qualities, should soon be a realistic possibility. lucosinolates - formerly t e r m e d t h e m u s t a r d oil glucosides - a r e amino acid-derived secondary p l a n t products t h a t contain a s u l p h a t e a n d a thioglucose moiety (Fig. 1), and occur in t h e order C a p p a r a l e s a n d a few o t h e r u n r e l a t e d fainilies of dicotyledons 1. T h e y are grouped into aliphatic, aromatic and indolyl glucosinolates b a s e d on w h e t h e r t h e y a r e derived from aliphatic amino acids (often methionine), phenylalanine or tyrosine, or tryptophan, respectively [or chain-elongated homologues t h e r e o f (e.g. homop h e n y l a l a n i n e and dihomomethionine)]. This d i v e r s i t y is f u r t h e r e x t e n d e d by secondary side-chain modifications such as hydroxylations, methylations, oxidations a n d des a t u r a t i o n s 2. A p p r o x i m a t e l y 100 different glucosinolates

G

© 1997 Elsevier Science Ltd

have been identified 2, 23 of which occur in A r a b i d o p s i s 3. Glucosinolates h a v e been detected in all organs of the plant, and are located w i t h i n t h e vacuole of the cell.

Glucosinolate degradation and the occurrence of degradation products in crops

The glucosinolate-myrosinase system The hydrolysis of glucosinolates is c a t a l y s e d by endogenous ~-thioglucosidases, the myrosinases, localized in t h e 'myrosin' cells s c a t t e r e d t h r o u g h o u t most p l a n t tissues. W i t h i n t h e s e cells t h e e n z y m e is stored inside m y r o s i n g r a i n s 4. The g l u c o s i n o l a t e - m y r o s i n a s e s y s t e m is a preformed two-component s y s t e m t h a t is a c t i v a t e d upon t i s s u e

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S--Glu NOSO3e

myrosinase SH + Glucose NOSO3°

RNCS (1) /

RSCN (III)

s

R=CHI

(v)

?Hz

O-.c/NH

RCN+ S (U)

II

S

(IV)

Fig. 1. Degradation of glucosinolates. After hydrolysis catalysed by myrosinase, the subsequent rearrangement of the unstable intermediate is dependent on side chains and hydrolysis conditions. The major degradation products are isothiocyanates (I), which are formed at pH>7, and nitriles (II), which are formed at pH<4. 2-propenyl-, benzyl- and 4-(methylthio)butylglucosinolates form thiocyanates (III). The presence of ~-hydroxylated side chains results in spontaneous cyclization to produce oxazolidine-2-thiones (IV). A terminal double bond in the side chain results in the formation of an epithionitrile (V), if an epithiospecifier protein and Fe2÷ions are present. Abbreviations: Glu, glucose; R, variable side chain.

damage. The hydrolysis products include thioglucose, sulphate and an unstable intermediate, which rearranges spontaneously to produce several degradation products, many of which have pronounced biological effects. Glucosinolates function as a defence against general herbivore attack and are implicated in host-plant recognition by specialized predators ~. The major degradation products include isothiocyanates, nitriles and thiocyanates, but epithionitriles and oxazolidine-2-thiones are also produced, depending on such factors as side-chain groups, pH and the presence of epithiospecifier protein 4'5 (Fig. 1). Epithiospecifier protein in combination with ferrous ions produces an epithionitrile when the glucosinolate contains a terminal double bond. Glucosinolates with ~-hydroxylated side chains spontaneously cyclize to yield the oxazolidine-2thiones. Distribution among crops Glucosinolates are present in several crop species, including oilseed rape (Brassica napus), where the presence of harmful degradation products restricts the amount of rape meal that can be used in animal feed supplements 6. In 426

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particular, isothiocyanates of 2-hydroxy-3-butenylglucosinolates (progoitrin), which account for up to 80% of total glucosinolates in rapeseed, undergo spontaneous cyclization to produce goitrin (5-vinyl-oxazolidine-2-thione), which may cause goitre among animals fed with rape meal. Other isothiocyanates, particularly methylsulphinylalkyl isothiocyanates, have been shown to induce anti-carcinogenic phase II enzymes such as quinone-reductases and glutathione-S-transferases ;. This suggests that high consumption of Brassica species (e.g. cauliflower, cress, brussels sprouts, cabbage and broccoli) could reduce the risk of developing cancer 7. The distinct flavour of glucosinolate-containing vegetables and condiment crops (e.g. mustard) is primarily caused by isothiocyanates. Isothiocyanates are partially volatile and play an important role as general repellents of microorganisms, insects and slugs s. However, some specialized pests of Brassicaceae are attracted to the volatile isothiocyanates, and are thought to use these compounds as feeding and oviposition stimuli ~. No primary physiological role has been assigned to glucosinolates, although it has been suggested that glucosinolate-bound sulphur is remobilized under conditions of sulphur deficiency9. Other studies, however, indicate that glucosinolates are unlikely to be a major source of recyclable sulphur 1°. The occurrence of anti-nutritional compounds in oilseed rape meal has led to efforts to reduce the natural level of glucosinolates in this species. Classical breeding techniques have resulted in an eight- to tenfold reduction in the content of aliphatic glucosinolates. An additional reduction and tissue-specific elimination or modification is desirable and could lead to significant crop improvement. This might be accomplished by genetic engineering aimed at the expression of regulatory or biosynthetic enzymes, but none of the genes responsible for glucosinolate biosynthesis has yet been isolated. Previous in vivo studies using seedlings or excised tissues have shown that amino acids, N-hydroxyamino acids, nitro compounds, oximes, thiohydroximates and desulphoglucosinolates are precursors of glucosinolates 11. Recent progress has been made in the characterization of key steps in the glucosinolate pathway, both by establishing biosynthetically active in vitro enzyme systems that catalyse the conversion of amino acids to oximes, and by identifying genes that regulate the variation in aliphatic glucosinolates.

Side-chainelongation The biosynthesis of glucosinolates comprises three independent stages (Fig. 2). First, the synthesis of chainelongated amino acids occurs. Next, the glucone common to all glucosinolates is added, and finally the side-chain modifications take place. The chain-elongated amino acids are probably derived from amino acids from proteins via a transamination reaction to produce the corresponding c~-keto-acids. This is followed by condensation with acetyl-CoA, and then a second transamination to recover the amino acid group (similar to the conversion of valine to leucine). Biochemical evidence for this scheme is based on the formation of chainelongated glucosinolates after in vivo administration of amino acids and acetate to plants Is. Although homo-, dihomo- and trihomomethionine-derived 2-propenyl-, but-3enyl- and pent-4-enylglucosinolates are the most common glucosinolates, trace levels of glucosinolates derived from chain-elongated methionine homologues [where R = CH~S(CH2)~ (see Fig. 1)] have been identified, with n as high

reviews

GSL on.s Metldonine

GSL-ELONG homo-methionine

~

GSL-ELONG dihomo-methionine

~

~homo-methionine

_

side chain elongation

fo,m~n of g~cone moiety

3-methyllhiopropyl GSL

1 o,,..ooo 3-methylsulphinylpropyl GSL

2-hydroxypropyl GSL

2-propenyl GSL

4-methy~iobutyl GSL

5-methylthiopentyl GSL

i

1

4-methylsulphinylbutyl GSL

3-butenyl GSL

GSL-OH 2-hydroxy-3-butenyl GSL

5-methylsulphinylpentyl GSL

side chain modification

4-pentanyl GSL

~ GSL-OH 3-hydroxy-4-pentenyl GSL

Fig. 2. A genetic model for the biosynthesis of aliphatic glucosinolates. Abbreviations: GSL, glucosinolate; QTL, quantitative trait locus. Courtesyof R. Mithen.

as 11. The genetic variation in the side-chain length of aliphatic glucosinolates has been studied in oilseed rape and Arabidopsis 13. A single locus, Gsl=elong, was shown to regulate the length of the aliphatic side chain and possibly also the total amount of aliphatic glucosinolates (Fig. 2). The role of Gsl-elong in determining side-chain length is supported by the observation that ecotypes of Arabidopsis that only contain propylglucosinolates have elong- alleles 14. Arabidopsis has a relatively simple genome, and has become the model plant for cloning genes by map-based approaches. A map-based cloning strategy has been used to isolate the Gsl-elong gene from Arabidopsis and to identify a YAC (yeast artificial chromosome) clone that hydridizes to RFLP (restriction-fragment length polymorphism) markers close to the gene 14. When cloned, the sequence may be used to identify Brassica homologues, or directly to manipulate the aliphatic glucosinolate profile in oilseed rape.

Development of the glucone moiety Conversion of amino acids to oximes

The first committed step in the formation of the glucone moiety of glucosinolates is the amino acid-to-oxime conversion. In independent studies in several species, the characterization of enzymes that catalyse this reaction has provided evidence that different enzymes are involved. The use of [UJ4C]-labelled tyrosine and phenylalanine has revealed that these amino acids are converted to the corresponding oximes by microsomes isolated from seedlings of, respectively, Sinapis alba (containing p-hydroxybenzylglucosinolate) and Tropaeolum majus (containing benzylglucosinotate) 15'16. The enzyme systems were shown to be cytochrome P450 monooxygenases by photoreversible carbon monoxide

inhibition and other inhibitor studies 1~'1~. These results provided the first experimental evidence of similarity between the enzymes that catalyse the conversion of amino acids to oximes in the biosynthetic pathway of glucosinolates and of cyanogenic glucosides, a related group of secondary plant products that likewise have amino acids as precursors and oximes as intermediates 17. It has therefore been proposed that homologous enzyme systems catalyse the conversion of amino acids to oximes in the glucosinolate and cyanogenic glucoside pathways 11. The biosynthetic pathway of cyanogenic glucosides has been elucidated using a biosynthetically active microsomal preparation isolated from etiolated seedlings of Sorghum bicolor, which produces the tyrosine-derived cyanogenic glucoside dhurrin 17. In the biosynthesis of dhurrin, a single, multifunctional cytochrome P450 enzyme catalyses the conversion of tyrosine to the corresponding oxime in a reaction that involves two consecutive N-hydroxylations, followed by a dehydration and a decarboxylation reaction is. Using microsomes isolated from young green leaves of oilseed rape, it has been possible to reveal the involvement of flavin-containing monooxygenases in the conversion of chain-elongated amino acids to their corresponding oximes ~9'2°. The process included an oxygen and NADPHdependent 14CO2release from [1J4C]-labelled homophenylalanine (the precursor of phenylethylglucosinolate) and dihomomethionine (the precursor of butenylglucosinolate). Gas chromatography in combination with mass spectroscopy indicated a simultaneous production of the oxime derived from homophenylalanine, but no mass spectrum could be obtained of the oxime from dihomomethionine because of instability 19. The lack of inhibition of the enzyme November1997,Vol. 2, No. 11

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reviews activities by carbon monoxide, cytochrome P450 inhibitors and antiserum raised against NADPH-cytochrome P450reductase indicated that the enzymes were not dependent on cytochrome P450s (Ref. 21). Inhibition of the activity by copper salts and diphenylene iodonium sulphate, and sensitivity to dithiothreitol, indicated that the enzymes were flavin-containing monooxygenases2°-22. Measurements of NADPH-dependent, oxidative decarboxylation were extended to include tri- and tetrahomomethionine 21. The three chain-elongated methionine homologues were all mutually competitive inhibitors of each other's monooxygenase activity. Furthermore, the methionine homologues were not inhibitors of the homophenylalanine monooxygenase activity. Based on these observations, it was concluded that at least two flavin-containing monooxygenases were involved in the biosynthesis of the chain-elongated aromatic and aliphatic glucosinolates in oilseed rape, one with substrate specificity for di- and trihomomethionine, and one with substrate-specificity for homophenylalanine. The monooxygenase profile in active preparations of microsomes generally corresponds to the spectz~um of glucosinolates present in a given species22. However, no activity could be detected using homomethionine [the precursor of sinigrin (2-propenylglucosinolate)] as substrate, even in species such as B. nigra where sinigrin is a major glucosinolate. Monooxygenase activities using dihomomethionine, trihomomethionine and homophenylalanine as substrates were found in all the species belonging to the Brassicaceae, including Sinapis spp. Interestingly, the Brassica spp. that contain p-hydroxybenzylglucosinolate did not have the corresponding tyrosine monooxygenase activity, but did have phenylalanine monooxygenase activity. In contrast, the Sinapis species that contain p-hydroxybenzylgiucosinolates had the cytochrome P450-dependent tyrosine monooxygenase activity, but no phenylalanine monooxygenase. It thus appears that Brassica species produce p-hydroxybenzylglucosinolate by p-hydroxylation of benzylglucosinolate, whereas the Sinapis spp. use tyrosine directly22. The conversion of tryptophan to indole acetaldoxime is the first step in the biosynthesis of both indolyl glucosinolates and the plant hormone indole acetic acid. The

Tyrosine

Phenylalanine ?

F;

conversion of tryptophan to indole acetaldoxime in seedlings of Chinese cabbage (Brassica carnpestris ssp. pekinensis) is catalysed by a plasma membrane-bound peroxidase 2~. Several plant species that do not contain glucosinolates have the same enzymatic activity, which suggests that the measured activity is related to the synthesis of indole acetic acid rather than to the biosynthesis of indolyl glucosinolares 24. In a comparative study it was shown that enzymatic activity involved in the formation of oximes from both chainelongated amino acids and from tryptophan in both oilseed rape and Chinese cabbage decreased with increasing maturity of the leaves 25. The peroxidase activity was present in all tissues, whereas the flavin-containing monooxygenase activities were absent from cotyledons and old leaves, as are the corresponding glucosinolates. The correlation between the distribution of biosynthetic activities and glucosinolates in leaves of oilseed rape and Chinese cabbage is in agreement with the indications of involvement of flavin-containing monooxygenases in the biosynthesis of chain-elongated aromatic and aliphatic glucosinolates, and peroxidases, in the biosynthesis of indolyl glucosinolates. From our current knowledge of the enzymes that catalyse the conversion of amino acids to oximes in the glucosinolate pathway, three different types appear to be involved: cytochrome P450s, flavin-containing monooxygenases and peroxidases (Fig. 3). Based on homology with the biosynthetic pathway of cyanogenic giucosides, the cytochrome P450-dependent monooxygenases in glucosinolate biosynthesis are believed to catalyse two consecutive N-hydroxylations followed by a dehydration and decarboxylation reaction TM. In the peroxidase-catalysed conversion of tryptophan to indole acetaldoxime, it has been proposed that the amino nitrogen is oxidized by H202, followed by dehydrogenation and decarboxylation reactions 24. No mechanism has been proposed for the reaction catalysed by flavin-containing monooxygenases. The involvement of three different enzymes in the conversion of amino acids to oximes suggests convergent evolution. From an evolutionary perspective, S. alba is an interesting species, because it contains a cytochrome P450-dependent monooxygenase for its tyrosine-derived glucosinolate; a peroxidase for its indolyl glucosinolates; and flavin-containing monooxygenases for the minor glucosinolates derived from chain-elongated Methionine Tryptophan amino acids. Interestingly, the cytochrome P450-dependent monooxygenases have only been identified in I chain extension I T. majus and Sinapis spp. [i.e. species I 1 I HMet DMet/TMet larger that contain only one glucosinolate (T. TetMet homol. majus) or one major glucosinolate among several minor forms (Sinapis I Per°xidasesl spp.)]. The evolutionary significance of this is presently unknown.

Oxirne Fig. 3. Summary of the different type of enzymes involved in the conversion of amino acids to oximes in the biosynthesis of glucosinolates. Abbreviations: P450, cytochrome P450; FP, flavin-containing monooxygenases; HMet, homomethionine; DMet, dihomomethionine; TMet, trihomomethionine; TetMet, tetrahomomethionine. Modifiedfrom Ref. 22 (courtesyof Roger Wallsgrove).

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Conversion of oxime to glucosinolate The conversion of oxime to thiohydroximate is a poorly understood step in the biosynthesis of glucosinolates, because no biochemical data are available on the intermediates involved. It has been proposed that the higher oxidation level of the thiohydroximate is obtained by oxidation of the oxime to an aci-nitro compound, which subsequently

reviews functions as the acceptor for an appropriate thiol donor 2~ (Fig. 4). In agreement with this proposal, incorporation of [14C]1-nitro-2-phenylethane (the tautomeric form of the acinitro compound) into benzylglucosinolate has been demonstrated in T. majus, although the incorporation rate was very low27. Furthermore, it was demonstrated in trapping experiments that l-nitro-2-phenylethane was formed in vivo from labelled phenylacetaldoxime. Conclusive evidence that the aci-nitro compound acts as an intermediate in the pathway is dependent on an in vitro demonstration of both its production from the oxime and its subsequent conversion to the thiohydroximate. The sulphur donor for the thiol sulphur is not known, although thioglucose has been excluded. Several inorganic and organic sulphur compounds have been incorporated into glucosinolates in vivo 27. In the plant, however, the sulphur compounds are interconvertable. Cysteine was the most efficiently incorporated sulphur source, suggesting that this compound is the immediate sulphur donor in the pathway under normal conditions. Conjugation of the thiol donor to the aci-nitro compound produces an S-alkylthiohydroximate (Fig. 4). The proposed conjugation of cysteine to the aci-nitro compound may be catalysed by a glutathione-S-transferase, as these enzymes are known to catalyse conjugation reactions to double bonds, and to substitute glutathione with cysteine. The Salkylthiohydroximate has been proposed to be cleaved by a C-S lyase to yield the thiohydroximate 28. A requirement for C-S lyase activity is that the substrate has an ~-hydrogen atom and a ~-thiol, which would be the case for an Salkylthiohydroximate produced as a cysteine conjugate. The final step in the development of the glucone pathway is an S-glucosylation of the thiohydroximate by a soluble UDPG:thiohydroximate glucosyltransferase to produce the desulphoglucosinolate, which is subseqLuently sulphated by a soluble 3'-phosphoadenosine 5'-phosphosulphate: desulphoglucosinolate sulphotransferase (Fig. 4). In contrast to the enzymes involved in the conversion of amino acids to thiohydroximate, both of these enzymes have been partially purified from several species. Characterization of a purified UDPG:thiohydroximate glucosyltransferase from oilseed rape has demonstrated that the enzyme has a high substrate specificity for thiohydroximates, but shows little specificity for the structure of the side chain 2~. Similarly, characterization of a sulphotransferase from Lepidium sativa has shown an absolute requirement for the desulphoglucosinolate structure, but no specificity for the structure of the side chain 3°. This lack of side-chain specificity appears to be a common feature for the enzymes involved in the last two steps in the pathway, and contrasts with the upstream enzymes involved in the conversion of amino acids to oximes. Side-chain modifications Secondary modifications of the side chains may occur following glucone development. In particular, the methioninederived aliphatic side chains are modified extensively (Table 1). Based on genetic studies, a model for side-chain modifications of aliphatic glucosinolates has been proposed31-33(Fig. 2). In spite of the great variation in aliphatic side-chain structures, these studies indicated that the diversity is the result of genetic variation in only three major loci: Gsl-oxid, Gsl-alk and Gsl-oh (Refs 31 and 32). The initial products after glucone formation are likely to be methytthioalkyl glucosinolates. Alleles at the Gsl-oxid

R-- CH-- COOH

!

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N

\

OH

oxime NADPH + O2 " ~

+

H

R--CH2--C/

II N®

G O / ~OH aci-nitro compound

S--CH2-- CH-- COOH

R-CH2-C / II N\

I

NH2

OH S-alkyl-thiohydroximate

SH R-CH2-C / II

N\ OH thiohydroximate

UDPG~

S--Glu

R-CH2--C / It

N\ OH desulphoglucosinolate PAPSq

S--Glu

R_CH2--C / II

N

\o-so glucosinolate

Fig. 4. A model for the biosynthesis of the glucone moiety of glucosinolates. There is currently no direct biochemical evidence for the boxed intermediates. Abbreviations: R, variable side chain; PAPS, 3'-phosphoadenosine 5'-phosphosulphate.

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locus result in the oxidation of the methylthiol group to methylsulphinylalkyl and methylsulphonylalkyl side chains. The Gsl-alk locus results in the production of alkenyl homologues by removal of the methylthio group, followed by insertion of a double bond. Gsl-ohp regulates the desaturation and addition of a hydroxy group to methylsulphinylpropyl glucosinolate, and is likely to be an allele of the Gsl-alk locus TM. The Gsl-oh locus regulates the hydroxylation of alkenylglucosinolates (except propenylglucosinolates) 33. Many of the alleles at the three loci are specific for side-chain length, which explains how these loci, in combination with the Gsl-elong alleles, can result in the complex diversity of glucosinolates. Biochemical studies have indicated that a cytochrome P450 monooxygenase catalyses the hydroxylation of 3-butenylglucosinolate to produce 2-hydroxy-3-butenylglucosinolate 34. A map-based cloning strategy has been used to identify a YAC clone that hybridizes to RFLP markers flanking the Gsl-alk locus TM. As the silique is the site of synthesis of aliphatic glucosinolates, antisense experiments in Brassica with the Gsl-alk gene driven by a silique-specific promotor could in principle prevent the formation of the 2-hydroxy-3-butenylglucosinolate, which is hydrolyzed into the major toxic compound (goitrin) in rapeseed meal 32.

Conclusion Significant progress has been made in recent years towards understanding the biochemistry and molecular genetics of the biosynthesis of glucosinolates. The indications are that three different enzymes catalyse the conversion of amino acids to oximes, suggesting that this step has evolved independently three times. The cytochrome P450 dependency of the oxime-producing enzyme in S. alba and T. majus indicates homology to the biosynthetic pathway of cyanogenic glucosides. The isolation of YAC and BAC (bacterial artificial chromosome) clones that hybridize to RFLP markers located close to the Gsl-elong and Gsl-alk genes suggests that the first Arabidopsis genes involved in the glucosinolate pathway will soon be available. These genes will be vitally important in attempts to initiate molecular strategies for modulating the level of glucosinolates in a tissue-specific manner in Brassica crops, with the aim of improving flavour and nutritional aspects.

Acknowledgements The authors are grateful to Dr Richard Mithen for contributing valuable information. Work was financed by the Danish Biotechnology Program. 430

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29 Reed, D.W. et al. (1993) Purification and properties of UDP-glucose: thiohydroximate glucosyltransferase from Brassica napus L. seedlings, Arch. Biochem. Biophys. 305, 526-532 30 Glendening, T.M. and Poulton, J.E. (1988) Glucosinolatebiosynthesis. Sulfation of desulfoglucosinolateby cell-free extracts of cress (Lepidium sativum L.) seedlings, Plant Physiol. 86, 319-321 31 Giamoustaris, A. and Mithen, R. (1996) Genetics of aliphatic glucosinolates. IV. Side chain modification in Brassica oleraceae, Theor. Appl. Genet. 93, 1006-1020 32 Mithen, R. et al. (1995) Genetics of aliphatic glucosinolates. III. Side chain structure of aliphatic glucosinolates in Arabidopsis thaliana, Heredity 74, 210-215 33 Parkin, I. et al. (1994) Genetics of aliphatic glucosinolates. II. Hydroxylationof alkenyl glncosinolates in Brassica napus, Heredity 72, 594-598 34 Rossiter, J.T., James, D.C. and Atkins, N. (1990) Biosynthesis of 2-hydroxy-3-butenylgiucosinolatesand 3-butenylglucosinolatein Brassica napus, Phytochemistry 29, 2509- 2512

Targeting of proteins into and across the thylakoid membrane Colin Robinson and Ale×andra Mant The assembly of the photosynthetic apparatus utilizes component proteins that are synthesized by two genomes and then targeted into and across the thylakoid membrane. The emerging picture is one of a remarkably complex system of protein trafficking, in which at least four distinct pathways operate within the chloroplast - two for lumenal proteins and two for integral membrane proteins. Some of the pathways can be traced back to the prokaryotic ancestor of the chloroplast, whereas others appear to have arisen more recently - one in response to the transfer of genes to the plant nucleus and another, possibly, in response to the acquisition of n e w photosynthetic proteins. Remarkably, proteins in three of these pathways are synthesized with cleavable signal-type peptides that are almost identical in overall structure, yet that execute entirely different functions. Recent studies have begun to reconcile the function of these targeting signals with the nature of the protein being targeted. he location, function and ancestry of the thylakoid m e m b r a n e have combined to make it an exceptional system in terms of protein targeting. The thylakoid is unique a m o n g known m e m b r a n e types in being a true internal membrane, and cytosolically synthesized proteins must therefore cross both the chloroplast envelope membranes and the stromal phase in order to reach their site of function. In the case of thylakoid lumen proteins, these m u s t be translocated across all three chloroplast membranes. Once targeted into the membrane, imported thylakoid proteins have to form complex p r o t e i n - p i g m e n t assemblies, an example of which is the photosystem II (PSII) complex

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depicted in Fig. 1. The P S I I complex contains over 20 polypeptides, among which are found extrinsic proteins on both the stromal and lumenal faces of the membrane, as well as a variety of single- and multispanning m e m b r a n e proteins. M a n y of these proteins are imported from the cytosol, and the targeting mechanisms for these m u s t be both precise and efficient. Finally, the entire system has had to adapt to a major upheaval as a consequence of chloroplasts evolving from endosymbiotic cyanobacterial-type prokaryotes. Whereas thylakoid proteins were once synthesized adjacent to their target m e m b r a n e in prokaryotes, the subsequent wholesale transfer of genes to the plant cell Ptl $1360-1385(97)01143-6

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