PHYSIOLOGIA PLANTARUM 97: 194-208. 1996Printed iit Denmark - att rights reserved

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Minireview

The myrosinase-glucosinolate system, its organisation andbiochemistry

Atle M. Bones and John T, Rossiter

Bones, A, M, and Rossiter, J. T. 1996. The myrosinase-glucosinolate system, its or-ganisation and biochemistry. - Physiol. Plant. 97: 194-208.

The myrosinase-glucosinolate system is involved in a range of biological activities af-fecting herbivorous insects, plants and fungi. The system characteristic of the orderCapparales includes sulphur-containing substrates, the degradativc enzymes myrosi-nases, and cofactors. The enzyme-catalyzed hydrolysis of glucosinolates initially in-volves cleavage of the thioglucoside linkage, yielding D-glucose and an unstable thio-hydroximate-C*-sulphcnate that spontaneously rearranges, resulting in the productionof sulphate and one of a wide range of possible reaction products. The products aregenerally a thiocyanate, isothiocyanate or nitrile, depending on factors such as sub-strate, pH or availability of ferrous ions, Glucosinoiatcs in crucifers exemplify com-ponents that are often present in food and feed plants and are a major problem in theutilization of products from the plants. Toxic degradation products restrict the use ofcultivated plants, e,g, those belonging to the Brassicaceae, The myrosinase-glucosino-late system may, however, have several functions in the plant. The glucosinolate deg-radation products are involved in defence against insects and phytopathogens, and po-tentially in sulphur and nitrogen metabolism and growth regulation. The compartmen-talization of the components of the myrosinase-glucosinolate system and the cell-spe-cific expression of the myrosinase represents a unique planl defence system. In this re-view, we summarize earlier results and discuss the organisation and biochemistry ofthe myrosinase-glucosinolate system.

Key words - Brassicaceae, Capparales, growth regulation, plant defence system,plant-phytopatogcn and insect interaction, sulphur and nitrogen metabolism.

A. M. Bones (corresponding author, e-mail atle.bones@unigen.ntnu.no). Unigen Cen-ter for Molecular Biology and Dept of Botany, MTFS, Univ. of Trondheim. N-7005Trondheim, Norway; arid J. T. Rossiter, Dept of Biological Sciences. Wve College.Univ. of London, Ashford, Kent. TN25 5AH. UK.

Introduction

Oilseed rape (Brassica napus L,) is a major Europeancrop, the most significant oil-producing crop of the tem-perate regions of central and northern Europe, The oil isvaluable in nutrition (low in polyunsaturates) and as anindustrial feedstock, for both specialist industrial useand as a potential fuel. The seed meal remaining after oilextraction is also a valuable animal feed, high in protein,which can be used in place of imported soybeans orother animal feed products. Many other species in theBrassicaceae are grown in Europe as human and animalfoodstuffs, cabbages, sprouts, kale, turnips, swedes, andmore, AU these species contain a group of secondarymetabolites termed glucosinolates. Upon mechanical

Received 27 April, 1995: revised 30 October, 1995

194

damage, infection or pest attack, cellular breakdown ex-poses the stored glucosinolates to degradative enzymes(myrosinases).

Plant breeding strategies over the past decade haveconcentrated on reducing the glucosinolate content ofrapeseed, to improve the acceptability of rapeseed mealand meet increasingly stringent requirements from theprocessing industry. It had earlier proved possible to re-duce the erucic acid content of rapeseed by breeding, toproduce the so-called 'single low' varieties. The low glu-cosinolate strategy has been successful, in that currentcommercial varieties ('double iow") have very low seedglucosinolate content, but there is a significant cost interms of crop protection and nutrition. Reducing or elim-inating the synthesis of specific glucosinolates, e,g, pro-

Physiol. Plant. 97, 1996

RNCS +(1)

S—Glc

OSO3'

myrcsinase

NOSO,-

- -f glc

RCN -f S + SO4^- + glc

(2)

Fig. I, The myrosinase/cofactcff catalyseddegradation ofglucosinolates to give (1)isothiocyanates, (2) nitriles,(3) cyanoepithioalkanesand (4) thiocyanates.

=-+ glc

R = allyl-, benzyl-,and 4-(methylthio)butyl-glucosinolates

RSCN + S04^

(4)

+ glc

goitrin, is a major goal in Brassica breeding. An altema-tive approach would be to change the amount of myrosi-nase available for hydrolysis of the glucosinolates.

The myrosinase-glucosinolate system has long beenthe defining phytochemical character of the order Cap-parales (Hegnauer 1986), This system and its compo-nents have been pre-eminent subjects of chemical inves-tigation of species within this order. Degradation of glu-cosinolates produces products that affect the value ofglucosinolate-containing plants when used as food forhumans or for feeding animals (Chew 1988), Thiocyan-ates can inhibit the uptake of iodine in the thyroid andlarge amounts are known to have negative effects on theliver. More recently, interest has focused on the potentialmutagenic, carcinogenic (Dunnick et al. 1982) and anti-carcinogenic (loannou et al, 1984) properties of natu-rally-occurring isothiocyanates.

The complexity of the myrosinase-glucosinolate sys-tem should indicate an important role in the life of thecruciferous plants. The function of this system may bediverse. The glucosinolates may be a sink for nutrientslike nitrogen and sulphur and the products of hydrolysismay have important roles in the defence system of theplants against microorganisms and insects. The numberof different glucosinolates and isoenzymes reported mayindicate that specific hydrolytic products are needed forcertain situations or developmental stages, Indoleglucos-inolates, e,g,, may be a sink for production of the planthonnone indoleacetic acid (IAA) and thereby be in-volved in growth regulation.

This review will focus on (1) how the glucosinolate-thioglucosidase system produces diverse hydrolysisproducts; (2) how the system is distributed and compart-mentalized; (3) how the system is held latent imtil the

products of the hydrolysis are needed; (4) what the prop-erties are of the myrosinases involved; and (5) what bio-logical function the myrosinase-glucosinolate system isinvolved in.

Glucosinolate hydrolysis products

The myrosinase-mediated degradation of glucosinolatesgives rise to an unstable thiohydroximate-O-sulphonatewhich, on release of sulphate (via a Lossen rearrange-ment), can result in the production of isothiocyanates,thiocyanates, nitriles and elementary sulphur, dependingon the concentration of H* and/or other factors (Fig, 1),Tbe mechanisms of degradation have been studied insome detail especially isothiocyanate and nitrile forma-tion (Benn 1977) although the mechanism for thiocyanatestill requires elucidation (Hasapis and MacLeod 1982),

Isothiocyanate and nitrile production

Isothiocyanates are usually produced at neutral pH whilenitrile production occurs at lower pH, Indole-glucosino-lates such as glucobrassicin undergo enzyme hydrolysisto give 3-indolemethanol, 3-indoleacetonitrile and 3,3'-diindolylmethane (Labague et al, 1991), The alcoholfrom the indole glucosinolates is derived from theisothiocyanate which undergoes solvolysis. Evidence foran indole isothiocyanate comes from work of Hanley etal, (1990) who isolated an indole isothiocyanate fromgluconeobrassicin degradation tmder specific experi-mental conditions, Isothiocyanates with a hydroxy groupin the 2 position spontaneously cyclise to give oxazoli-dine-2-thiones, an example of which is goitrin, derivedfrom 2-hydroxy-but-3-enylgIucosinolate,

Physiol. Plant. 97, 1996 195

Thiocyanate production

Of the naturally occurring glucosinolates, only three ap-pear to undergo enzymic degradation to thiocyanates.These are allyl-, benzyl- and 4-(methylthio)butylglucos-inolates. Various mechanisms have been proposed forthiocyanate formation and the most recent is a combinedtheory where an isomerase causes Z-E isomerisation ofthe aglycone but only glucosinolates with stable cationsare able to tindergo E-aglycone rearrangement to thethiocyanate (Hasapis and MacLeod 1982),

Epithioalkanes

Epithioalkanes are produced from the hydrolysis of alke-nyl glucosinolates when myrosinase co-occurs with asmall labile protein known as epithiospecifier protein(Tookey 1973),

The myrosinase enzyme systemOccurrence of myrosinases

Myrosinase and glucosinolates were first discovered inmustard seeds by Bussy (1840), For a historical reviewof myrosinase investigations see, e,g, Bjorkman (1976)and A, M, Bones (1991, Thesis, Univ, of Trondheim,Norway), The myrosinases always appear to be accom-panied by one or more glucosinolates. They occur in allBrassicaceae (Cruciferae) species examined and havealso been found in Akaniaceae, Bataceae, Bretschneider-aceae, Capparaceae, Caricaceae, Drypetes (Euphorbi-aceae), Gyrostemonaceae, Limnanthaceae, Morin-gaceae, Pentadiplantdraceae, Resedaceae, Saivadora-ceae, Tovariaceae and Tropaeolaceae (Rodman 1991),Enzymes with myrosinase activity have also been foundin the fungi Aspergillus sydowi (Reese et al, 1958, Oth-suru et al, 1969) and Aspergillus niger (Ohtsuru et al,1973), in the intestinal bacteria Enterobacter cloacae(Tani et al, 1974) and Faracolobactrum aerogenoides(Oginsky et al, 1965), in mammalian tissues (Goodmanet al, 1959) and in tbe cruciferous aphids Brevicorynebrassicae and Lipaphis erisimi (MacGibbon andBeuzenberg 1978), These aphid myrosinases hydrolyze2-hydroxy-2-phenyiethyl glucosinolate in vitro and ex-hibit electrophoretic mobilities distinct from those ofisoetizymes isolated from their host plants. This distinc-tive bitxhemical behaviour does not rule out the possi-bility that the myrosinases are modified from enzymesacquired from the host platit, in contrast to the de novosynthesis involved in endogenous myrosinases in fungiand bacteria. However, recent work has shown that anti-bodies raised against plant myrosinase do not cross reactwith partially purified aphid myrosinase, indicating thatthe protein is not derived from the plant host (M,Bridges, R, Cole, A, Ecoles Jones and J, F, Rossiter, un-published results).

The amount of myrosinase activity found in seeds ofcultivars of Sinapis alba L,, Brassica campestris L, and

Brassica napus L, has been examined by Henderson andMcEwen (1972), Bjorkman and Lonnerdal (1973) andBones (1990), The myrosinase activity was found to beabout 10 times higher in S. alba than in B. campestrisand the activity in fi, napus was slightly higher than inB. campestris (Bones 1990).

Myrosinase isoenzymesThe existence of multiple forms of myrosinase has beenshown in many plants. By analytical gel electrophoresisvarious authors have demonstrated the separation of sev-eral myrosinase isoenzymes (MacGibbon and Allison1970, Henderson and McEwen 1972, Buchwaldt et al,1986), MacGibbon and Allison (1970) examined theisoenzyme pattem after electrophoretic separation ofmyrosinases of 7 species of Rhoeadales, Each specieswas found to have a distinct pattem and the number ofdetectable bands varied from 1 to 4. They also found dif-ferent pattems depending on whether the extracts weremade from ieaf, stem, root or seed, Buchwaldt et al,(1986) reported that the crude extract from seeds of 5/-napis alba contained at least 14 myrosinase isoenzymes,Isoeiectric focusing in polyacrylamide gels of ethanol-powder preparations revealed 2 bands for Brassica ntgraand Brassica napus and 7 bands for Sinapis alba (Buch-waldt et al, 1986), In a study of the effects of ascorbicacid on myrosinases from different crucifers, Hendersonand McEwen (1972) noticed different activation of thevarious isoenzymes as detected by an increase in bariumsulphate precipitate.

The validity of the method for development of myro-sinase bands after electrophoretic separation should becontrolled, Henderson and McEwen (1972) stated that itwas possible to locate active isoenzymes in a Crambeabyssinica and two Brassica juncea preparations, eventhough activity could not be detected by a direct spectro-photometric method, A possible explanation for thisvery surprising result is that the conditions were changedas a result of the electrophoretic run. However, anotherpossibility may also be that this assay is not as specificas it should be. Only active myrosinase is detected inthis assay.

Recently, James and Rossiter (199!) separated twomyrosinases from cotyledons of 5-day-old Brassica na-pus seedlings. The most striking difference between thesetwo myrosinases was the degree of glycosylation, Myro-sinase I was similar to myrosinase C reported by Lonner-dal and Janson (1973) and Bones and Slupphaug (1989),while the myrosinase II was reported to be considerablyless glycosylated. By immunobiots with polyclonal anti-bodies against myrosinase and by periodic acid-Schiffstaining these two myrosinases were shown to be differ-ent. When tested for substrate specificity against 5 glu-cosinolates, the degree of activation by ascorbic acid wasalso different (James and Rossiter 1991),

Little is known about the substrate specificity of my-rosinase isoenzymes. The data available is from James

196 Physiol. PJant. 97. 1996

and Rossiter (1991) and Bjorkman and Janson (1973),The two myrosinases examined by James and Rossiterdegraded different glucosinolates at different rates.However, both isoenzymes showed highest activityagainst aliphatic glucosinolates and least activity againstindole glucosinolates. From their results it is evident thatmembers of a given class of glucosinolates are appar-ently degraded at approximately the same rate in vitro,an exception being the significant difference in degrada-tion of sinigrin. Similar results were also obtained byBjorkman and Janson (1973), who also noted that the ac-tivity against an aliphatic glucosinolate (progoitrin) wasdependent on the isoenzyme tested. The results from Ar-abidopsis thaliana could indicate that myrosinases ac-cept a wide range of glucosinolate substrates, Xue et al.(1992, 1995) and Chadchawan et al, (1993) have all re-ported that there are three myrosinase genes in Arabi-dopsis thaiiana. Active transcripts have only been re-ported for two of these genes, and they are supposed todegrade the 23 different glucosinolates reported to bepresent in Arabidopsis thaiiana (Haughn et al, 1991),When considering substrate specificity one should alsobe aware of the possibility that the specificity could beaffected by associated factors like epithiospecifier pro-tein, myrosinase-binding protein or other myrosinase as-sociated proteins or components. To clarify the questionof substrate specificity it will be necessary to examineisoenzymes from different developmental stages in com-bination with the known associated factors.

Distribution of myrosinase activityDistribution of the myrosinase isoenzymes seems to beboth organ- and species-specific, Electrophoretic exami-nation of isoenzymes from many plants, organs or tis-sues demonstrates that the distinct pattems may varywith species, organ and age of the plant (MacGibbonand Allison 1970, Henderson and McEwen 1972, Buch-waldt et al, 1986), Little is known about the physiologi-cal reason for this difference. It has been postulated thatthe particular isoenzymes correspond to endogenousconditions found in that plant, or to conditions fotind inthe target organism, or to particular glucosinolates thatdominate the proftle of that tissue. It is not knownwhether all these bands after electrophoretic separationrepresent real isoenzymes with a distinct amino acid se-quence. As a consequence more effort should be placedon determining whether or not these are isoenzymes, Acombination of immunological detection, amino acid se-quencing and characterization of myrosinase genes willbe used to solve this problem.

There have been few reports with a systematic analy-sis of the variation in myrosinase activity in plants at dif-ferent developmental stages and in different organs.Bones (1990) examined the myrosinase activity at dif-ferent deveolopmental stages and in different parts of theplant in a full life cycle, A comparison between plantsregenerated from protoplasts, explants and seed-germi-

nated plants was also included. The results (Bones 1990)show that hypocotyls contain the highest specific myro-sinase activity of the seedling organs. The specific activ-ity in hypocotyls was always more than twice that in cot-yledons and normally several times that in seedlingroots. Although the highest speciftc myrosinase activitywas observed in hypocotyls, cotyledons contain mostmyrosinase activity based on total aaivity. But since thetotal protein content in cotyledons was several times theamount in roots and hypocotyls the specific activity waslower (Bones 1990), In a study of the myrosinase ex-pression in Sinapis alba L,, Xue et al, (1993) detectedmyrosinase mRNAs in cotyledon, hypocotyl, root andleaves of 6-, 8- and 14- day-old seedlings. Highest levelsof myrosinase mRNA accumulation were observed forhypocotyls at day 6 and 8 and in leaves at day 14 aftergermination.

The reported activities in different tissues varies, but alltested organs/tissues contain some myrosinase activity(Bones 1990), Myrosinase activity could also be detectedin callus cultures and in plants cultured in vitro. Exceptfor the roots of fully grown plants where a high activitywas observed, organs of mature plants normally containedlow myrosinase activity (Bones 1990), References to sev-eral other studies of myrosinase activity can be found inBones (1990), James and Rossiter (1991) reported that thetotal potential myrosinase activity increased during earlyseedling growth, when the activity was measured in thepresence of 0,3 mM ascorbic acid.

Using a slot-blot technique, Falk et al, (1992) showedthat myrosinase mRNA also was produced in all testedorgans of B, napus. Xue et al, (1993) have shown thatmyrosinase mRNA in seedling organs decreases duringearly seedling growth, Xue et al, (1993) examined thetranscript levels from members of two subfamilies ofmyrosinase genes in developing seedlings of Sinapisalba. One of the myrosinase genes (MB=Myr2) wastranscribed in all organs of the seedlings, while the othermyrosinase gene (MA=Myrl) showed no transcription inthe investigated organs of the seedling. Expression ofboth MA and MB myrosinase genes was observed duringembryo development of Sinapis alba.

To examine the possible correlation between myrosi-nase and myrosin cells the myrosinase activity in calluswas measured and an ultrastmcturai study performed.Although low, a significant myrosinase activity wasfound. In common with other observations of low myro-sinase activity, no myrosin cells could be detected withconventional LM- and TEM-techniques, It was stressedthat lack of observable myrosin cells seemed to be fol-lowed by a low myrosinase activity, indicating a positiverelationship between the enzyme and the myrosin cells(Bones 1990),

Springett and Adams (1989) investigated the distribu-tion of myrosinase in Bmssels sprouts (B. oleracea L, var,bullata subvar, gemmifera). Myrosinase activity was 4-5times higher in the outer leaves of the Brussels sproutsthan in other regions (stalk, inner leaves and centre).

Physiol. Plant. 97,1996 197

Tab, 1. Physico-chemical characteristics of selected purified myrosinases.', Bones and Slupphaug (1989):', Lonnerdal and Janson (1973);', Biorkman and Lonnerdal tl973): ••, Biorkman and Janson {1972): •, Ohtsuru and Hata (1972); , Ohtsuru and Kawatani (1979), ND, not', Bjorkman and Lonnerdal (1973): \ Bjorkman and Janson (1972);determined.

Parameter

Molecular mass (kDa)Number of subunitsSubunit mass (kDa)Carbohydrates (%, wAv)Isoeiectric point (pi)Km (sinigrin) ()iM)pH optimumTemp, optimum (°C)Ascorbic acid (]iM)optimum activationMetal activationMetal inhibition

Brassica napus'

1542

779,6/11,7/18,95,0/4,96/4.94

68/71/725,475

400 (2,8/3,0/3,8x)NDND

Brassica napus^'

1352

6514

4.96/4,99/5.0630

4,560

700 (40x)NDND

Sinapis alba'

1512

6218

5,081705,860

700 (4x)NDND

Mustard seed^

1524

4015,8/17.8/22,5

4,6NDNDND

ActiveNDND

Wasabi japonictf

48012

45^7NDND470

6,5-7,037

ActiveCa-"/Mn='Cu=*/Hg-*

Mjrosinase isolation and puriflcation

Myrosinase isoenzymes from plant sources show sub-stantial diversity in physico-chemical characteristics(Tab, 1), All myrosinases isolated and purified so farhave been reported to be glycoproteins. The carbohy-drate content varies from 9 to 23% of the total molecularmass, James and Rossiter (1991) purified a myrosinasefrom 5-day-old Brassica napus seedlings with low levelsof glycosylation. Molecular mass of purified myrosi-tiases normally range from 125 to over 150 kDa, One ex-ception is the myrosinase from Wasabia japonica, whichwas reported to have a molecular mass of 580 kDa (Oht-suru and Kawatani 1979), Isoeiectric points vary be-tween 4,6 and 6,2 (Bjorkman 1976),

Several reports have described the isolation and char-acterization of myrosinase from white mustard (Bjork-man and Janson 1972, Bjorkman and Lonnerdal 1973,Buchwaldt et al, 1986) and oilseed rape (Bjorkman andLotmerda! 1973, Lonnerdal and Janson 1973, Buchwaldtet al, 1986, Bones and Slupphaug 1989), Ohtsuru andHata (1972) purified four myrosinase isoenzymes frommustard powder; three had a molecular mass of 153 kDa,and the fourth 125 kDa, From SDS-polyacrylamide gelelectrophoresis the number of subunits was estimated tobe 4 (Tab, 1), Bjorkman and Janson (1972) purified onemyrosinase to apparent homogeneity and obtained a par-tial purification of two other isoenzymes. The completelypurified myrosinase was shown to contain 2 subunits ofsimilar size, A new purification procedure for myrosi-nase from B. napus L, was presented by Bones andSlupphaug (1989), When comparing the results it shouldbe kept in mind that there are few reports of apparentlyhomogeneous myrosinase purifications, Lenman et al,(1990) reported that myrosinases could form complexeswith molecular masses in the range 140-800 kDa,

Effects of ascorbic acid

Ascorbic acid has been shown to modulate myrosinaseactivity in some species (Nagashima and Uchiyama1959), A model for the action of ascorbic acid was pos-

tulated by Tsuruo and Hata (1968), Ascorbic acid doesnot participate in the reaction catalyzed by mustard my-rosinase (Ettlinger et al, 1961, Tsuruo and Hata 1968),nor is it involved in the association of the enzyme sub-units (Ohtsum and Hata 1973), The activation appears tobe due to a conformational change in the protein struc-ture, leading to an enhanced reaction rate when the ef-fector binding sites are occupied (Tsuruo and Hata 1968,Ohtsum and Hata 1973), Tsuruo and Hata (1968) postu-lated the presence of one site of action for the substrateand two for the ascorbic acid. The substrate site has twomoieties, one for the glycone and one for the aglyconepart of the glucosinolate. The conformation of the agly-cone moiety is altered when the ascorbic acid site is oc-cupied, so that the glucosinolate fits better in its site. Be-cause the second site for ascorbic acid is the same as thesubstrate site, high concentrations of ascorbic acid havean inhibitory effect. When /)-nitrophenol-/J-D-glucoside(p-NPG) is used as a substrate high concentrations ofascorbic acid inhibit the reaction due to competition atthe binding site. Moderate concentrations have no effect,since p-NPG does not occupy the aglycone moiety of theglucosinolate site. Considering that a weaker associationof glucosinolates to the myrosinase may give an in-creased reaction rate, this may also explain the increasedK^ in the presence of ascorbic acid. When ascorbic acidis added both the K^ and the W^^ are increased. De-creasing the affinity of the enzyme for the substrate is notthe normal behaviour of a positive effector. However, aspostulated by Tsumo and Hata (1968) this may be due toa common binding site for the effector and the substrate.

The lack of activation of p-NPG hydrolysis in thepresence of ascorbic acid has, however, been questionedrecently (Durham and Poulton 1990), A 2,6-fold activa-tion of hydrolysis of p-NPG was found in the presenceof 1 mM ascorbic acid using principally the same meth-odology. If this is correct, the model of activation byascorbic acid has to be reevaluated. The possibility ex-ists that only the glucose and sulphate group of the sub-strate is involved in binding to the active site, and thatthe R-group is outside the binding site, and contributingonly by electrostatic effects with the areas surrounding

198 Physiol. Plant. 97, !996

the binding site. This would also imply that the confor-mational change is in the active site,

Ohtsuru and Hata (1973) investigated the inhibition ofmyrosinase from brown mustard by trinitrobenzene-sulfonic acid (TNBS) in the presence and absence ofascorbic acid. In the presence of 1 mM ascorbic acid andTNBS, myrosinase activity was reduced by 55% whileTNBS alone gave no inactivation. These results supportthe theory that the activation mechanism of myrosinaseby ascorbic acid depends on a slight conformationalchange of the protein, and is not due to the dissociationand association mechanism of myrosinase, L-Ascorbicacid has been reported to be solely localized in vacuoles(Grob and Matile 1980, Matile 1980),

Myrosinase binding proteins

Several polypeptides are associated with myrosinases byvirtue of their co-precipitation with anti-myrosinase an-tibodies (Lenman et al, 1990), However, it is not knownat this stage if they have a role in the myrosinase-glucos-inolate system and the biochemistry of the phenomenonhas yet to be fully explored. The function of thesepolypeptides are unknown, but it has been shown thatwounding induces the expression of these polypeptides(Falk et al. 1995), A series of ongoing experiments willprobably yield more information about the proteinsbound to or associated with myrosinase,

Falk et al, (1995) characterized the myrosinase-bind-ing proteins (MBP) first observed by Lenmati et al,(1990), At least 10 proteins in extracts of Brassica napusreacted with a monoclonal antibody against MBP, Theproteins were classified as myrosinase-binding proteins(MBP) and myrosinase-binding protein-related proteins(MBPRP), MBPRP were present in almost all parts ofthe piant, whereas MBP were exclusively found inseeds. Two MBP with molecular masses of 50 and 52kDa, respectively, and two MBPRP with molecularmasses of 80 and 100 kDa, respectively, were puriftedand characterized and found to display stmctural simi-larities. Based on differences observed in, e,g,, solubil-ity, Falk et ai, (1995) suggested that they could have dif-ferent functions, Falk et al, (1995) hypothesised thatthey could have some role in response to tissue damagesince the myrosinase-glucosinolate system is activatedupon tissue damage, Lenman et al, (1990) concludedthat the possibility exists that MBP50 and 52 are identi-cal to epithiospecifier protein (ESP), The lack of relationbetween the ESP activity (J, T, Rossiter, M, Bridges andF, H, Ling, unpublished result) and expression of MBP(Falk et ai, 1995) does not support that ESP are similarto MBP The distribution of MBP and MBPRP in differ-ent species is not known. The function, cellular, and sub-cellular localization of MBP and MBPRP so far remainspeculative. Another group of myrosinase-associatedproteins (MAP) with afftnity for myrosinase complexeshas recently been found (A, Falk, personal communica-tion). These polypeptides are based on amino acid se-

quence information and immuno-reactivity utirelated tothe MBP and MBPRP polypeptides.

Epithiospecifier protein (ESP)

Epithiospecifier protein is a small protein (30-40 kDa),first isolated from Crambe abyssinica seeds (Tookey1973), This protein does not have myrosinase activity,but it interacts with myrosinase to promote sulphurtransfer from the S-glucose moiety to the terminal alke-nyl moiety (Fig, 1), Degradation of progoitrin (2-hy-droxy-3-butenyl glucosinolate) in the absence of ESPproduces mainly oxazolidine-2-thione. The same degra-dation in the presence of ESP produces mainly epithion-itrile, ESP is unique in that it specifies the reaction prod-ucts and not the substrate. The function of ESP in vivo isunknown. The effects of epithionitriles in mammalshave been evaluated by, e,g,, Wallig et al, (1988) andVan Steenhouse et al, (1991),

Formation of epithionitriles represents an altemativepathway for alkenyl glucosinolates with terminally unsat-urated carbon (e,g,, progoitrin, sinigrin, gluconapin andglucobrassicanapin). This degradation step is mediated byESP and the conditions under which the thioglucosidecleavage occurs (MacLeod and Rossiter 1987), Combina-tions of myrosinase and ESP from various plant sourcesproduced the same products, demonstrating that epithioni-triles are formed from glucosinolates by the same mecha-nism in all crucifers tested (Petroski and Tookey 1982),Similar results were also obtained with the myrosinase-producing fungus Aspergillus sydowi (Petroski andKwoiek 1985), Cole (1975) investigated the distributionof aglucones upon autolysis of glucosinolates in 8-week-old plants in 79 species, Cyano-epithioalkanes were de-tected in 15 of these 79 species indicating the presence ofESP, Species with ESP activity include Crambe abyssin-ica (Tookey 1973), Atyssum perenne, A. saxatile, Arabi-dopsis thaliana, Berteroa incana, Brassica chinensis, B.juncea, B. napus, B. oleracea, B. rapa, Cakile maritima,C. pratensis, Hirchfetdia incana, Lepidum sativum, Lubu-taria maritima, Sisymbrium altissimum and Turritis gla-bra (Cole 1975, MacLeod and Rossiter 1985),

Kinetic studies show that epithiospecifier protein in-hibits myrosinase activity non-competitively (MacLeodand Rossiter 1985), providing evidence that ESP inter-acts at a site on the myrosinase other than the substratebinding site. Ferrous ions are essential to epithionitriieformation (MacLeod and Rossiter 1985), Epithiospeci-fier protein in fi. rmpus was shown to be inactive in theabsence of ferrous ions (Macleod and Rossiter 1985),Addition of ferrous ions changed the major products ofthe hydrolysis from oxazolidine-2-thione to epithioni-triie.

Genes encoding myrosinase

A significant amount of work has been put into the clon-ing of myrosinase genes. As a result, several myrosinase

Physiol. PtMt. 97, 1996 199

genes from Sinapis alba, Brassica napus and Arabidop-sis thatiana have been isolated and characterized (Falk etal, 1992, 1995, Xue et al, 1992, Chadchawan et al, 1993,Thangstad et al, 1993), The results show that myrosi-nases in Brassica napus are encoded by a multigene fam-ily consisting of three subgroups (Thangstad et al, 1993,Falk et al, 1995), Thangstad et al, (1993) have suggestedthat the subgrotips should be named according to the re-commendations of the Commission on Plant Gene No-menclature (Plant Mol, Biol, Reporter 11; 3-9, 1993),

The first cDNA gene encoding myrosinase was iso-lated from Sinapis alba by Xue et al, (1992), The genewas isolated from an immature seed cDNA library byscreening with degenerated oligonucleotides obtainedfrom amino acid sequence information. One full-lengthclone of the Myr2-xype (MB3) was isolated with a probeobtained from a polymerase chain reaction based on se-quence information from a truncated clone of the Myrl-type, that was fn-st isolated (MAI). The clone MB3 con-sisted of 1878 bp, started with a 118-bp 5'untranslatedregion and had a 20-amino-acid signal sequence. Theclones were grouped into MA(Myrl)- and MB(Myr2)-type based on their sequence similarity, MB(Myr2)clones showed 92-94% sequence identity, whereasMA(Myrl) and MB(Myr2) showed 62-64% similarity.These data are based on only one full-length cDNAclone, cDNA encoding myrosinases from Brassica na-pus and Arabidopsis thaliana have also been reported(Falk et al, 1992, Chadchawan et al, 1993), The fi, napusgene was found to be 91,2% identical to the MB3 clonefrom Sinapis alba and 67,5% identical to the MAI clone,at the amino acid level, and therefore to belong to theMyr2 subtype (=MB) of the gene family.

Five functional genomic myrosinase genes have so farbeen sequenced, two from Brassica napus (Thangstad etal, 1993), two from Arabidopsis thaliana (Xue et al,1995) and one from Brassica campestris (Machlin et al,1993), The first two genomic myrosinase genes that werecharacterized, MyrLBnl and Myr2.Bnl, from Brassicanapus, are about 3 kb in length, with 11 small introns and12 exons (Thangstad et al, 1993), The homology is83,2% at the amino acid and 77,4% at the nucleic acidlevel (Thangstad et al, 1993), A signal peptide and a vari-able number of sites for N-linked glycosylation indicatetransport and glycosylation through the ER-Golgi com-plex. Thangstad et al, (1993) concluded that Myrl.Bnlmost likely encodes a major seed myrosinase, and thatMyr2Bnl encodes a vegetative type myrosinase, A se-quence comparison of the five functional myrosinase ge-nomic genes has been presented by Xue et al, (1995),

A myrosinase pseudogene has also been reported(Lenman et al, 1993), The pseudogene was found tospan more than 5 kb, A portion of the 5'-end was miss-ing and it contained probably 12 exons. The uncertaintywas due to missing exon-intron splice sites. Sequencecomparisons with myrosinase cDNA grouped this geneto the Myrl (MA) subgroup of the gene family, A multi-ple sequence aligntnent with the cDNA from Falk et al.

(1992) and several other jj-glycosidases and ^-galactosi-dases revealed that myrosinases belonged to the (BGA)family of /S-glycosidases (Lenman et al, 1993),

Myrosinases are encoded by a large gene family inboth Sinapis alba and Brassica napus, Thangstad et al,(1993) estimated the number of myrosinase genes in B.napus at 14—17, A sitniiar result was also reported byLenman et al, (1993) who estimated that there were 13Myr2 (MB) genes and 3-4 MyrJ(MA) genes in Sinapisalba. Some of these genes may be pseudogenes (Len-man et al, 1993), Present results indicate that these esti-mates may be too low (V, BeisvSg, P, Winge and A, M,Bones, unpublished results; Falk et al, 1995), In Arabi-dopsis thaliana a small myrosinase gene family withonly three genes has been found (Chadchawan et al,1993, Xue etal, 1995),

Falk et al, (1992) found highest expression of myro-sinase mRNA in young leaves, cotyledons and develop-ing seed, in which it was detected from day 15 to day 30after pollination. Later, Xue et al, (1993) reported thattwo myrosinase genes show a tissue preferential expres-sion during embryo and seedling development in Sina-pis aiba. Northem blots with mRNA from seeds andyoung leaves of Brassica napus probed with MA (Myrl)and MB(Myr2) specific probes, showed that the twosubgroups of the myrosinase gene family were differen-tially expressed (Lenman et ai, 1993), The highest levelof expression of MA(Myrl) was found in seeds,whereas MB(Myr2) expression was found to be highestin leaves. The large number of genes which are tran-scriptionally active indicate that the myrosinase-glucos-inolate system must play an important role in the life ofthe piants of Brassicaceae (O, P, Thangstad, P, Wingeand A, M, Bones, unpublished results).

Gene structure

Based on conserved regions in cDNA from three spe-cies, polymerase chain reaction (PCR) primers weremade, and used to amplify and characterize the stmctureof the myrosinase genes in Brassica napus, B. chinensis(B. campestris var, chinensis), B. campestris, B. olera-cea, Cheiranthus cheiri, Raphanus sativus and Sinapisalba. The strong similarity of nucleotide sequence, in-tron-exon structure and gene copy number between theseven species compared, indicate that the myrosinasegenes are similarly organised in these species. Work inour laboratory (P, Winge and A, M, Bones, unpublishedresults) shows that the structure of myrosinase genes inArabis atpina, Tropaeolum majus, Iberis umbellata andLepidum sativum is similar to that reported by ThangstadetaL(1993),

Myrosin cells

The term myrosin cell was ftrst used by Guignard (1890)and has later been used to describe this special type of celldiscovered by Heimicher (1884) and assumed to contain

200 Piiysiol. Plant. 97, 1996

myrosinase, Myrosin cells were confined to parenchyma-tous tissue of the green parts of different plants of theBrassicaceae, especially epidermal cells of leaves (for re-view of distribution, occurrence, morphology reported inearly studies see Bones and Iversen 1985),

The importance of the myrosin cells as a taxonomictool has been generally accepted and noted by numerousauthors (see Dahlgren 1980, Jorgensen 1981), Jergensen(1981) used occurrence and distribution of myrosin cellsas two of several criteria for the classification of the or-der Capparales,

As a consequence of the taxonomic investigationssome information about the occurrence and distributionof myrosin cells was generated, Myrosin ceils were ob-served in seeds, parenchyma tissue, epidermis, andguard cells (for references see Bones and Iversen 1985),

The first reports on the morphology of myrosin cellswere based on light microscopic observations. Althoughthe resolution obtained in these studies was limited,some interesting observations were reported, Spatzier(1893) observed that the grains in myrosin cells differedfrom those in surrounding cells. The grains in myrosincells had a different refractivity and were more homoge-neous. Therefore he suggested the term myrosin grains.The myrosin grains did not have inclusions (globoids),had a higher refractivity and gave an intense reactionwith general protein stains.

Later reports on the structure of myrosin cells havemainly used the higher resolution obtained in the elec-tron microscope. Rest and Vaughan (1972) followed thedevelopment of protein bodies and oleosomes(spherosomes) during 9 stages of embryo developmentin Sinapis alba L, Special attention was paid to the cellsin the developing cotyledons and the accumulation ofproteins in the protein bodies. The myrosin cells couldbe distinguished from the aleurone celis 24 days afterpetal fall. At this stage the central vacuole has subdi-vided. In aleuron cells proteins were first observed aselectron dense lumps with globoids. The initial accumu-lation of protein in myrosin cells was reported to includevacuolar structures which had more irregular outlinesand were ftlled with a homogeneous fibrillar material(Rest and Vaughan 1972),

Bones and Iversen (1985) presented an analysis of thedistribution of myrosin cells in plants of Raphanus sati-vus and Sinapis alba. In seeds of Raphanus sativus L,and Sinapis alba L,, imbibed for 2 h, the relative area oc-cupied by myrosin cells was in the range 0,9-1,6% and4,5-6,5%, respectively (Bones and Iversen 1985),Twenty-ftve days after seed set, the identification of my-rosin cells was speculative and solely dependent on thedifferent reaction after staining.

The morphology of myrosin cells varies and dependson the organ and tissue in which they are present as wellas the age of the tissue (Bones and Iversen 1985), In cot-yledons, two morphologically distinct types of myrosincelis have been observed. In palisade tissue of cotyle-dons elongated tnyrosin cells were frequently observed.

In the parenchyma tissue both elongated and isodiamet-ric cells were observed (see, e,g.. Bones and Iversen1985), In roots and hypocotyls myrosin cells are gener-ally found in an elongated fortn in longitudinal sections.Compared to normal cortical cells they are 1-6 times thesize of surrounding cells,

Myrosin cells in seeds and young seedlings are char-acterized by homogeneous protein bodies, in contrast toprotein bodies in aleurone-like cells that usually containelectron opaque globoids. In the electron microscopemyrosin grains appear moderately electron dense andwith a finely granular content (Bones and Iversen 1985),Changes in the ultrastmcture of myrosin cells of Sinapisalba and Raphanus sativus have been followed system-atically both during early seedling growth and embryodevelopment (Bones and Iversen 1985), The myrosingrains of myrosin cells in hypocotyls and roots changedramatically during the first days after soaking. The my-rosin grains undergo an active period which first seemsto include division of the protein bodies and later fusionsto fortn the big centra! vacuole.

Dilated dstemae of the endoplasmic reticulum

Several attempts have been made to correlate the pres-ence of dilated cistemae of the endoplasmic reticulumwith myrosinase (see references in Bones et al, 1989),Evidence for such a correlation has not been presented,

Compartmentation of components in the myrosinase-glucosinolate system'The myrosinase-giucosinolate bomb'

Matile (1980) concluded that the stability of glucosino-lates in the intact root tissues of horseradish (Armoraciarusticana) appeared to be due to the location of glucosino-lates and myrosinase in distinct subcellular compartments

Tonoplast

Plasmalemma

Amyloplast

Mitochondfioti

Fig, 2, The mustard oil bomb, A model for compartmentation ofmyrosinase, glucosinolates and ascorbate proposed by LUthyand Matile (1984), * Indicate suggested localization of myrosi-nase. II should be stressed that myrosinase now has been shownto be localized in protein bodies/vacuoles of the idioblastsnamed myrosin cells (shown in Fig, 3),

Physiol. Ptant. 97, 1996 201

of the same cell. Evidence was presented for the presenceof myrosinase in extracellular compartments (cell walls)and associated with the cytoplasmic side of intemal mem-branes, while giticosinolates were localized in vacuoles,,A.ctivation of this system would be induced by permeabi-lization of membranes resulting in the release of glucosi-nolates and L-ascorbic acid from the centra) vacuoles ofparenchyma cells, Iti a later report from Luthy and Matile(1984), a rectified analysis of tiie subcellular organisationof the myrosinase system was presented. The main differ-ence between the model of Matile (1980) and the modelpresented by Llithy and ,Vlatile (1984), was that myrosi-nase in the latter report was reported to be a cytosoiic en-zyme which tended to bind to membranes. This rectified'Mustard bomb'-ntodel is presented in Fig, 2. Luthy atid,Matile (1984) concluded that the association of myrosi-nase with smooth ER, plasmaiemma, dictyosomes andmitochondria as reported by Iversen (1970), could reflecttrue localization of myrosinase in situ.

Cellular and subcellular localization of niyrosinase

Attempts to localize myrosinase in plants have now beenreported for more than 100 years and involve either mor-phological, anatomical and liistochemical ob,ser\ ations orcytochemical, ceil fractionation and biochemical studies.Using the former approach, several workers have consid-ered mvrosinase to occur in myrosin cells that stain spe-cifically with ,V[illori's reagent, orcein solution and con-centrated hydrochloric acid, and iactophenol aniline blue(see Rest and Vaughan 1972, Bones and Iversen 1985).Staining techniques have demonstrated that the grains inmyrosin cells and aleurone-like cells are of differentchemical origin. However, since no histochemical re-agents were fotind that reacted specifically with eithermyrosinases or glucosinolates, a definitive conclusionabout the nivrosin cell content could not be made.

The first attempt to localize myrosinase at the cellularlevel was reported by Guignard in 1890. Since thenmany attempts have been made that have given strik-ingly different results (Peche 1913, Iversen 1970, Pi-ha"kaski and Iversen 1976, Matile 1980, Liithy and Mat-ile 1984), Techniques which have been u,sed lo localizemyrosioase have included light- and electron-micros-copy and a combination of microscopy and detection ofenzyme activity (Iversen 1970) or microscopy cotnbinedwith histochemical stains (Bones and Iversen 1985),cell- and subceilolar fractiotiatioti (Fihakaski andIversen 1976, Luthy and Matiie 1984), organelle separa-tion bv centrifugation techniques, comparison of thenumber of myrosin cells with the detectable tnyrositiaseactivity, metnbrane fractionation and purification of my-rosin eel! protoplasts (Bones and Iversen 1985),

The first localization based on a cytochemical methodwas reported by Peche (1913), The method employed wasbased on enzymatic hydrolysis of the substrate sinigrin,which produced sulphate in sitti on fresh sections ofRaphanus sativus. By including BaCl, in the substrate a

precipitate of insoluble BaSO,, was deposited at the site ofthe enzyme reaction. This cytochemical method showedthat myrosinase could be localized in myrosin cells,iversen (1970) concluded tliat most of the myrosinase ac-tivity was confined to the dilated cislemae of the endo-plasmic reticulutn and in a limited extent to the mitochon-dria. Using the same cytochemical technique, Maheswariet al, (1981) concluded that myrosinase was largely asso-ciated with the plasmalemma of most cells, Luthy andMatile (1984) u,sing subcellulat fractionation, suggestedthat most of the myrosiria,se activity was associated withthe tonoplast, plasmalemma and endoplasmic reticulum,and that although niyrosinase was preseni in the cytosol ithad a remarkable tendency to adhere to membrane sur-faces, Matile (1980) suggested that myrosinase was a cy-tosolic enzynie. while the glucosinolates are compartmen-talized in the vacuoles. Pihakaski and Iversen (1976) con-cluded that the myrosinase activity is mainly found in dic-tyosomes and stiiooth endoplasmic relictilum.

The initial light- and electron tiiicroscope investiga-tions indicated a connection between myrosin ceils andmyrosinase. However, evidence for the localizationcould not be obtained solely on the basis of microscopy.The development of techniques based on specific anti-bodies opened up the possibility of performing ati immu-nohistocheinical locaUzation of myrosinase. The combi-nation of knowledge of myrosin cells, their distributionand specific antibodies, finally enabled the localizationof myrosinases in myrosin ceils (Thatigslad et ai. 1990),Further immunogold-EM studies proved the subcellularlocalization of myrosinase in the protein bodies/vacuolesof myrosin cells (Thang,stad et al, 1991). Examples ofimmunocytochemical and immunogold-EM localizationof myrosinase are shown in Fig, 3, The cellular and sub-cellular localization was verified by Hoglund et al,(1992), The possibility that minor amounts of myrosi-nase may be present in other compartments is still open(Thangstad et al, 1991), The cell and tissue specific lo-calization of myrosinase has also been verified by in situhybridization experiments with subfamily-specific tny-rosinase probes (Xue et ai, 1993),

As shown by Bones et al, (1991) myrosin cells do un-dergo considerable developmental changes during thefirst 14 days after seed set. This seems to include a dra-matic change in the compartmentatioti due to a reorgani-zation of the protein bodies/vacuoles (Bones et al, 1991).

Localization of giucosinolates and ascorbic acid

Glucosinolates and ascorbic acid have been reported tobe localized in vacuoles of non-specific cells (Grob andMatile 1979, Matiie 1980, Helmitiger et al, 1983), Asshown by seyeral authors (e.g., Nagashima andUchiyama 1959, Ettlinger et al, 1961, James and Ros-siter 1991) the degradation rate of glucosinolate in-creases considerably in the presence of ascorbic acid.Grab and Matile (1980) examining root tissue of Ar-moracia rusticana, found that 99,5% of the ascorbic acid

202 Phvsiol. Hani. 97. !'>%

Fig. ,̂ . Examples ol celltilar (A-D) and suhccllLild! iLl localization ot mxrosiaa^e NKiosinasc show.s tcU spccihc expie^.sion (.A-D), Differential interference contrast miLio>,cop\ (A,Ci and ilic same scetums empkning lmmumieUivheiiiieal lahellini; x\iili anti-myrosina.se antilx>die.s (B,D) ,shows that in_M(»Mnast, is e\plc^seli HI nsMossn ^clls (arrows in .\ and C shtn\ eeUs fluorcscinc in Band D). .\l the subcellular level myr<isinase is Lompartmontalised in pnilem hodies/\aciioies (EinaineJ mvrosin sraiiis iMGi. B,ir: 2Mm.

was compartmentilized in vacuoles. In samples of roottissue they measured an ascorbic acid concentration ofapproximately 2,0 mM. Considering that neariy all theascorbic acid was localized in the vacuoles, the concen-tration of ascorbic acid in this organeile should be con-siderably higher.

Organi,sation of the myrosinase-jiiueosinolatf ,systeni

The probletn of the cellular localization of glucosino-lates still remains unsolved. Since glucosinolates are hy-drolyzed only after the plant is injured, at least the fol-lowing three alternatives are possible for the location of

Phvsjol. Plant. »>7, 19% 20,"?

substrate and enzyme, respectively: (1) in different cells;(2) in different compartments of the same cell; and (3)inside the same compartment of the same cell, but in aninactive form,

Myrosinase, glucosinolates and ascorbic acid may re-side in the same compartment, the myrosin grains/vacu-oles of myrosin cells. High concentrations of ascorbicacid inhibit and low concentrations activate myrosinaseactivity (Bjorkman 1976), Optimal ascorbic acid con-centration for myrosinase from Armoracia rusticana hasbeen reported to be 1,8 mM (Ohtsuru and Hata 1979),Together with the value for the total ascorbic acid con-centration in Armoracia rusticana of 2,0 mM as reportedby Grob and Matile (1980), this can be considered tosupport the hypothesis suggested above. If ail ascorbicacid is compartmentalized in vacuoles, disruption of thetonoplast membranes should give a maximum activationof the myrosinase and therefore a maximum response af-ter wounding. As discussed in Bones and Slupphaug(1989), ascorbic acid is an unusual activator. It inhibitsat high concentrations and activates at low concentra-tions. In a system such as that just described, this wouldbe an ideal effector molecule. Based on the results ofGrob and Matile (1980), the ascorbic acid concentrationin vacuoles of Armoracia rusticana must be consider-ably higher than 2,0 mM. At this concentration of ascor-bic acid the myrosinase enzyme system would be inac-tive. The co-localization of myrosinase and glucosino-lates would be most likely if this is a defence system. Inthis case, a co-localization makes the system a 'toxicmine', which could be activated simply by disruption ofthe tonoplast membranes. After disruption the ascorbicacid concentration will drop due to dilution and myrosi-nase will be activated,

A localization of glucosinolates and myrosinase indifferent vacuoles in the same cell is not likely. The vac-uoles of a myrosin cell do undergo considerable changesafter sowing (Bones et al, 1991) of which the fissionsand fusions of the vacuoles in the cells are the most re-markable. To maintain the stability of such a system, ahitherto utiknown mechanism for sorting and fusion ofglucosinolate or myrosinase containing vacuoles is nec-essary, A more likely system would include compart-mentation of glucosinolates in vacuoles of some cellsand myrosinases in vacuoles of myrosin cells. The dem-olition of subcellular compartmentation by mechanicaldisruption or by micro- or macro-organisms feeding onthe plants would cause the necessary contact betweenenzyme and substrate.

Another possiblity is that myrosinase is localized inmyrosin cells and other components of the system inseparate cells. To activate such a system enzymes orsubstrate must be transported or the organisation of a tis-sue disrupted.

The three sub-groups of myrosinases recently re-ported (Thangstad et al, 1993, Falk et al, 1995) may alsohave relevance to the organisation of the enzyme sys-tem. The two genes belonging to the Myrl and Myr2

subgroups of the gene family were reported to have 3and 9 potential glycosylation sites (Thangstad et al,1993), Since glycosylation may be a requisite for a vacu-olar localization due to the hydrophilic properties addedwith the carbohydrates, this could indicate that myrosi-nase also can be localized in other places than the vacu-ole/myrosin grain.

From the abovementioned it can be concluded that thecellular organisation of the myrosinase-glucosinolatesystem is unclear. Evidence shows that myrosinases,glucosinolates and ascorbic acid are localized in vacu-oles, but with the exception of myrosinase which is lo-calized in myrosin cells, the cellular localization of theother components including myrosinase associated pro-teins are unknown.

Responses to plant microbe and pest interactions

Mechanical wounding and infestation of oilseed rape(Koritsas et al, 1991) with cabbage stem flea beetle(Fsylliodes chrysocephala) have been shown to induceindole glucosinolate biosynthesis, while the aliphaticglucosinolates decreased. Similar effects have also beennoticed in leaves of oilseed rape (Doughty et al, 1991)and roots (Birch et al, 1990) challenged with the dark-leaf-spot pathogen (Aiternaria brassicae) and the fieldslug (Deroceras reticutatum), respectively.

Work in our laboratories has shown that infection withfungi can induce a local synthesis of myrosinase (V,Karapapa, J, Heale and J, T. Rossiter, unpublished re-sults). The possibility exists that other stress responsesalso induce a similar response. Initial results did not re-veal any increase in total myrosinase activity, but thiscould be due to a high constitutive synthesis of myrosi-nase.

Plant growth hormones from glucosinolates?

In most higher plants de nova indoleacetic acid (IAA)biosynthesis proceeds ffom L-tryptophan via indoiepyru-vic acid or tryptamine to indoleacetaldehyde, which isfinally oxidized to IAA (Schneider and Weightman1974), Species of Brassicaceae have a complex indolemetabolism where IAA biosynthesis may follow differ-ent pathways. In Brassicaceae, indole-3-acetaldoxime ismetabolized to indolemethylglucosinolate (glucobrassi-cin) and several of its derivatives which have beenshown to accumulate in the vacuole (Helmlinger et al,1983), The presence of in vivo conversion of indole-3-methylglucosinolate has been discussed, and it has beenestablished that over the pH range from 4 to 7 indole-3-acetonitrile (IAN) may be formed by enzymatic degra-dation of indole-3-methylg!ucosinolate by myrosinase invitro (Searle et al, 1982), One possibility is therefore thatthe indole glucosinolate pool can act as a sink for pro-duction of indoleacetic acid. By a step to IAN the indoleglucosinolates can be converted by a nitrilase (Bestwicket al, 1993) to IAA which in addition to its normal hor-

204 Physiol. Plant. 97, 1996

mone action may also be involved in the response actionafter, e,g,, infestation, i,e, a part of the defence system ofthe plant. Four Arabidopsis thaliana cDNAs encodingnitrilases with key roles in the biosynthesis of IAA haverecently been cloned (Bartel and Fink 1994),

Sulphur and nitrogen and efTects on glucosinolateand myrosinase levels

Since glucosinolates contain a significant proportion ofsulphur and nitrogen it might be expected that fertiliserswill influence the concentrations of glucosinolates inBrassica crops. It has been suggested that under condi-tions of sulphur deficiency sulphur bound in glucosino-lates of Brassica species can be remobilised by enzy-matic cleavage with myrosinase (Schnug et ai, 1993),The mechanism for this is thought to involve the controlof myrosinase activity by the ascorbate/glutathione cycle.Studies have shown (Schnug et al, 1995) a close relation-ship between sulphur status and glucosinolate concentra-tions and glutathione although inter-dependencies forascorbate are less apparent.

Field work with Brassica carinata, B. Juncea and fi.napus (examining irrigation and nitrogen levels) hasbeen carried out and quality parameters such as proteinand sinigrin content measured (Singh et al, 1994), Irriga-tion had no effect on these parameters although in-creases of nitrogen, up to 120 kg N h a ' , increased sini-grin content. Work by Fieldsend and Milford (1994a)with single- and double-low cultivars of oilseed rapedemonstrated large differences in the ability to synthe-sise glucosinolates and showed these differences to berelated to developing pods rather than vegetative tissues.Sulphur measurements showed that the glucosinolatescontained only a small proportion of the crops sulphurand were unlikely to be a major source of recyclable sul-phur even under conditions of severe sulphur deficiency,Glucosinolate profiles (Fieldsend and Milford 1994b)have been determined throughout the life cycle of fouroilseed rape cultivars (Bienvenue, Ariana, Cobra andCapricorn) at defined stages of development. It wasshown that substantial differences developed in the pro-files of these compounds during vegetative growth.Changes in the profiles of glucosinolates throughout theplant's development are thought to have implications forpests and diseases,

A decreasing sulphur supply to the plants results in adecrease in free sulphate and glucosinolate concentra-tions and an increase in myrosinase activity (Underhiil1980, Schnug 1990), This implies that the increase inmyrosinase activity during sulphur stress could have thefunction of a remobilization of sulphate sulphur fromglucosinolates, because sulphate and isothiocyanates canbe utilized as sulphur sources in the primary metabolismof the plant (Machev and Schraudolf 1978), Sulphatehas recently been shown to induce differential expres-sion of myrosinases (Bones et al, 1994), This representsa hitherto unkown mechanism of activation by an inor-

ganic compound, A possible function of the sulphate in-duction may be that an initial degradation caused by me-chanical disruption of the tissue (and thereby the my-rosin cells), releases sulphate from degradation of glu-cosinolates which again signals that more myrosinase(and glucosinolates?) is needed.

It appears that various environmental stresses will af-fect myrosinase levels and glucosinolate concentrationsand composition and more work is required to estabUshthe underlying biochemical mechanisms that control thisbiosynthesis,

Isoforms with different functions?

It has been shown that the expression of specific myrosi-nases can be both organ specific (James and Rossiter1991, Xue et al, 1993, Bones et al, 1994) and that somecan be induced by specific components like sulphate(Bones et al, 1994) or jasmonic acid (L, Rask, personalcommunication), James and Rossiter (1991) discussedthe existence of two myrosinases in 5-day-old cotyle-dons, one glycosylated and one witb low giycosylation. Itwas suggested that the glycosylated myrosinase could beinvolved in the defence system, because the enzyme islocated throughout the seedling, and because it was twiceas active in degrading indoie glucosinolates as was thenon-glycosylated myrosinase (myrosinase II), This ex-planation is supported by the results presented by Korit-sas et al, (1991). They showed that the infestation ofadult B. napus plants by the cabbage stem flea (Psyl-liodes chrysocephala L.) resulted in an accumulation ofindoie glucosinolates in damaged tissues.

It has been shown for B. napus and three other cruci-fers that while total glucosinolate levels drop during earlyseedling growth, endogenous ascorbate concentration in-creases (Sukhija et al, 1985), Furthermore, James andRossiter (1991) have shown that the total "potential" my-rosinase activity in seedlings, i,e, the enzyme activity inthe presence of 0,3 mM ascorbate, increases during day2-6 after sowing. This may indicate that myrosinase H isinvolved in hydrolysis of aliphatic glucosinolates, Glu-cosinoiates can perhaps be considered to be a storage ofreserve compounds that may be mobilized in the devel-oping seedlings, A better knowledge of the sub-cellularlocalization of the compounds involved would makethese interpretations easier.

Conclusions and future research

There is an increase in the interest for the tnyrosinase-glucosinolate system. This is at least partially becausesome of the tools that are necessary for molecular investi-gation of the system have now been developed. Futureresearch will focus on different topics of the system ofwhich some will be briefly mentioned here.

The physiological role of glucosinolates will be inves-tigated in detail. These studies will probably include ex-periments that will detennine the role of glucosinoiates

Physiol. Plant. 97, 1996 205

for growth of the plants including the role at differentstages such as the vegetative growth period, genninationand flowering. There are already some indications thatdouble-zero oilseed rape is more sensitive to sulphur de-fiency than single-zero plants. Although there have beenattempts to correlate the levels of glucosinolates with,e,g,, growth and resistance, future experiments should bemore specific and determine the direct effect of a lowlevel of indole-glucosinolates on specific growth periodssuch as flowering.

The myrosinase sequence data available show thatthere are at least three subgroups of the myrosinase genefamily in Brassica napus. This may indicate that the dif-ferent genes are involved in different processes in theplant. Information from expression studies using probesfrom different genes have already shown that myrosi-nases can have a tissue speciftc expression (Xue et al,1993), Transformation of plants containing myrosinaseand also plants which do not contain myrosinase will beused to study the effects of over-expression and under-expression (anti-sense). Localization of the glucosino-lates and the myrosinase associated proteins at a sub-cel-lular level will help greatly in the work to determine thefunctions of the system.

The goal will be to understand the molecular mecha-nisms and thereby obtain a better understanding of bio-logical processes where the myrosinase-glucosinolatesystem is involved. Another goal will be to use these re-sults in breeding programs. The amount, distributionand mixture of specific glucosinolates are targets forbreeding programs. For example, high concentrations ofglucosinolates in parts of plants that are not used forfeed. Modification of plants to obtain the optimal com-bination of myrosinases and glucosinolates is a finalgoal. The results wanted will probably differ and de-pend on where the plants are to be used. Control ofcompounds as insect repellants and attractants willprobably be important, because a decline in the amountof plant pesticides which can be used in the future is ex-pected. However, the glucosinolate-myrosinase systemconsists of more than 100 substrates and several en-zyme forms. To engineer this system specific informa-tion about its components and the genes involved isneeded, A combination of traditional plant breeding andgenetic engeenering could possibly produce some of thewanted traits.

Acknowledgments - Research in our groups has been supportedby the Norwegian Research Council (A. B.) and by the Agricul-ture and Food Research Council and British Council (3. T, R),

References

Bartel, B. & Fink, G. R. 1994, Differential regulation of anauxin-producing nitrilase gene family in ArabidopsisIhaliana. - Proc. Natl, Acad, Sci. USA 91: 6649-6653.

Benn, M, 1977. Glucosinolates, - Pure Appl, Chem, 49: 197-210,

Bennett, R, N. & Wallsgrove, R. M, 1994, Secondary metabo-lites in plant defence mechanisms. Tansley Review No. 72. —New Phytol. 127:617-633.

Bestwick, L. A., Gronning, L, M,, James, D. C Bones, A, &Rossiter, J, T. 1993. Purification and characterization of anitrilase from Brassica napus. - Physiol, Plant. 89: 811-816,

Birch, A. N. E., Griffiths, D, W, & MacFarlane Smith, W. H.1990, Changes in forage and oilseed rape (Brassica napus)root glucosinolates in responce to attack hy tumip root fly(Delia fioralis). - 3. Sci, Food Agric. 51: 309-320.

Bjorkman, R, 1976, Properties and function of plant myrosi-nases, - In The Biology and Chemistry of Cruciferae (J, G.Vaughan, A, J. MacLeod andB, M,G. Jones, eds), pp. 191-205, Academic Press, London, ISBN 0-12-715150-8,

- & Janson, J, C, 1972, Studies on myrosinases. I. Purificationand characterization of a myrosinase from white mustardseed (Sinapis alba L,), - Biochim. Biophys, Acta 276: 508-518,

- & Lonnerdal, B. 1973. Studie.s on myrosinases. III, Enzy-matic properties of myrosinases from Sinapis alba and Bras-sica napus seeds. - Biochim, Biophys, Acta ,327: 121-131,

Bones, A, M. 1990. Distribution of/?-thicglucosidase activiiy inintact plants, cell and tissue cultures and regenerated plantsof Brassica napus L. - J, Exp. Bot, 41: 737—744,

- & Iversen, T.-H, 1985. Mvrosin cells and myrosinase. - Isr,J.Bot. 34: 351-375.

- & Slupphaug, G. 1989. Purification, characterization andpartial amino acid sequencing of /J-thioglucosidase fromBrassica napus h.-i. Plant Physiol, 134: 722-729,

- , Evjen, K. & Iversen, T,-H. t989. Characterization and dis-tribution of dilated cistemae of the endoplasmic reticulum inintact piants, protoplasts, and calli of Brassicaceae. - Isr. J.Bot. 38: 177-192,

- , Thangstad, O. P,, Haugen, O. A. & Espevik, T 1991. Fateof myrosin cells: Characterization of monoclonal antibodiesagainsi myrosinase. - J, Exp. Bot. 42: 1541-1549,

- , Ponnampalam, S. & Thangstad, O. P 1994. Sulphate caninduee differential expression of thioglucoside glucohydro-lases (myrosinases). - Pianta 193: 558-566,

Buchwaldt, L., Larsen, L, M,, Pioger, A. & Sarensen, H, 1986,Fast polymer liquid chromatography isolation and character-ization of plant myrosinase, /f-thioglucoside glucohydrolase,isoenzymes. - J. (ihromatogr 363: 71-80.

Bussy, A, 1840. Sur la formation de I'huile essentielle de mou-tarde. - J . Pharm, 27: 464-^71.

Chadchawan, S., Bishop, J., Thangstad, O, P,, Bones, A. M.,Mitchell-Olds, T & Bradley, D, 1993. Arabidopsis cDNAsequence encoding myrosinase, - Piant Physiol, 103: 671-672.

Chew, F. S, 1988a, Biological effects of glucosinolates. -In Bi-ologically Active Natural Products for Potential use in Agri-culture (H. G. Cutler, ed.), pp, 155-181. The AmericanChemical Society, Washington, DC.

- I988h. Searching for defensive chemistry in the Cruciferae,or, do glucosinolates always control interactions of Cru-ciferae with their potential herbivores and symbionts? No!(K, A, Spencer, ed,), pp. 81-112, Chemical mediation of co-evolution. Academic Press, San Diego, CA,

Cole, R. A. 1975, Cyanoepithioalkanes: nitriles and thiocyanateas products of autolysis of glucosinolates in Cruciferae, -Phytochemistry 15: 759-762,

Dahlgren, R, 1980. A revised system of classification of the an-giosperms. - Bot, J. Linn. Soc. 80: 91-124,

Doughty, K. J,, Porter, A, J, R., Morton, A, M,, Kiddle, G.,Bock, C. H, & Wallsgrove, R. 1991. Variation in the glucos-inolate content of oilseed rape (Brassica napus L,) leaves, ILResponse to infection by Aiternaria brassicae (Berk,) Sacc,- Ann, Appl, Biol, 118:469-^77,

Dunnick, J. K,, Prejean, J, D,, Ha,seman, J,, Thompson, R, B.,Giles, H, D. & McConnel, E, E. 1982. Carcinogenesis bioas-say of allyl isothiocyanate. - Fundam, Appl, Toxicol. 2:114-120.

206 Physiol. Plant. 97, 1996

Durham, P, L, & Poulton, J, E. 1990, Enzymic properties of pu-rified myrosinase from Lepidium sativum seedlings, - Z,Naturforsch, 456: 173-178,

Ettlinger, M, G,, Dateo, G, P, Harrison, B. W,, Mabry, T. J. &Thompson, C. P. 1961. Vitamin C as a coenzyme: The hy-drolysis of mustard oil glucosides, - Proc, Natl. Acad, Sci.USA 47: 1875-1880.

Falk, A,, Xue, J,, Lenman, M. & Rask, L. 1992. Sequence of acDNA clone encoding the enzyme myrosinase and expres-sion of myrosinase in different ti.ssues of Brassica napus. -Plant Sci. 83: 181-186.

- , Ek, B, & Rask, L, 1995a, Characterization of a new myro-sinase in Brassica napus. - Plant Mol, Biol, 27: 863-874.

- , Taipalensuu, J., Ek, B., Lenman, M. & Rask, L. 1995b,Characterization of rapeseed myrosinase-binding protein. -Pianta 195:387-395.

Fieldsend, J, & Milford, G, F, i. 1994a, Changes in glucosino-lates during crop development in single- and double-lowgenotypes of winter oilseed rape (Brassica napus). I, Pro-duction and distribution in vegetative tissues and developingpods and potential role in recycling of sulphur within thecrop, -Ann, Appl. Biol. 124: 531-,542.

- & Milford, G, F, J. 1994b. Changes in glucosinolates duringcrop development in single- and double-low genotypes ofwinter oilseed rape {Brassica napus). II, Profiles and tissue-water concentration in vegetative tissues and developingpods.-Ann. Appl, Biol. 124: 543-555,

Fenwick, G, R., Heaney, R, K. & Mullin, W. J, 1983. Glucosino-lates and their breakdown products in food and food plants.-CRCCrit, Rev, Food, Sci. Nutr. 18: 123-201,

Goodman, I,, Fouts, J, R., Bresnick, E,, Menegas, R, & Hitch-ings, G. H, 1959. A mammalian thioglycosidase. - Science1,30:450-451.

Grob, K. & Matile, Ph. 1979. Vacuolar location of giucosino-lates in horseradish root cells. - Plant Sci. Lett. 14: 327-335,

- & Matile, Ph, 1980. Compartmentation of ascorbic acid invacuoles of horseradish root cells. Note on vacuolar peroxi-dase. -Z . Pflanzenphysiol, 98: 235-243.

Guignard, L. 1890. Recherche.s sur la localisation des principesactifs des Cruciferes. - J. Bot. 4(22): 38,S-395.

Haughn, G, W,, Davin, L,, Giblin, M. & Underhiil, E. W. 1991.Biochemical genetics of plani secondary metabolites in .Ara-bidopsis thaliana. The glucosinotates. - Plant Physiol. 97:217-226.

Hanley, B, A., Parsley, K. R., Lewis, J. A. & Fenwick, R. G.1990. Chemistry of indole giucosinolates: Intermediacy ofindole-3-ylmethyl isothiocyanates in the enzyme hydrolysisof indole glucosinolates. - J, Chem. Soc. Perkin Trans. 1:2273-2276.

Ha,sapis, X, & MaCleod, A. J, 1982. Benzylglucosinolate degra-dation in heat-treated Lepidium .lativum seeds and detectionof a thiocyanate-forming factor, - Phytochemistrj' 21: 1009-1013,

Hegnauer, R, 1986. Phytochemistry and plant taxonomy, - Anessay on the chemotaxonomy of higher plants. - Phy-tochemistry 25: 1519-1,535.

Heinricher, E, 1884. Uber Eiweissstoffe fuhrende Idioblasten beieinigen Cruciferen, - Ber. Dtsch. Bot, Ges. 463-̂ 167.

Helmlinger, J., Rausch, T. & Hilgenberg, W. 1983. Localizationof newly synthesized indole-3-methylglucosinolate (=gluco-brassicin) in vacuoles from horseradish (Armoracia rusti-cana). - Physiol, Plant, 58: 302-310,

Henderson, H. M. & McEwen, T, J, 1972. Effect of ascorbicacid on thioglucosidases from different crucifers. - Phy-tochemistrj II: 3127-3133.

Hoglund, A. S,, Lenman, M. & Rask, L, 1992, Myrosinase is lo-calized to the interior of myrosin grains and is not associatedto the surrounding tonoplast memhrane, - Plant Sci, 85:165-170.

loannou, Y,, Burka, L, T. & Matthews, H, B, 1984, Allyl isothio-cyanate: comparative disposition in rats and mice. - Toxicol,Appl, Pharmacol. 75: 17,3-181,

Iversen, T,-H, 1970, Cytochemical localization of myrosinase

(/(-thioglucosidase) in root tips of Sinapis alba L, - Proto-plasma 71: 451-466.

James, D, & Rossiter, J, T, 1991, Development and characteris-tics of myrosinase in Brassica napus during early seedlinggrowth, -Physiol. Plant. 82: 163-170.

JSrgensen, L. B, 1981, Myrosin cells and dilated cistemae of theendoplasmic reticulum in the order Capparales. — Nord. J.Bot, 1: 433-445,

Koritsas, V. M,, Lewis, J, A, & Fenwick, G, R. 199!, Glucosino-late responses of oilseed rape, mustard and kale to mechani-cal wounding and infestation by cabbage stem flea beetle(Psvlliodes chrvsocephala). - Ann, Appl. Biol. 118: 209-221'.

Labague, L,, Gardrat, G., Coustille, J. L,, Viaud, M. C, & Rollin,P. 1991, Identification of enzymatic degradation productsfrom synthesised glucobrassicin by gas chromatography-mass spectrometry. - J. Chromatogi. 586: 166-170.

Lenman, M,, Rodin, M,, Josefsson, L.-G. & Rask, L. 1990. Im-munological characterization of rapeseed myrosinase. — Eur,J, Biochem, 194:747-753.

- , Falk, A,, Xue, h & Rask, L. 1993, Characterization of aBrassica napus myrosinase pseudogene: myrosinases aremembers of the BGA family of /?-glycosidases, - Plant Mol,Biol, 21: 463-474.

Liithy, B. & Matile, Ph. 1984. The mustard oil bomb: Rectifiedanalysis of the subcellular organisation of the myrosinasesystem. -Biochem. Physiol, Pflanz. 179: 5-12,

Lonnerdal, B, &. Janson, J.-C. 1973, Studies on m\Tosinases. ll.Purification and characterization of a myTosinase from rape-seed {Brassica napus L.). - Biochim, Biophys, Acta 315:421-429.

MacGibbon, D, B. & Allison, R. M. 5970. A method for the sep-aration and detection of plam glucosinoiases (myrosinases),- Phytochemistr) 9: 541-544.

- & Beuzenberg, E. J. 1978. Location of glucosinolase inBrevicorvne brassicae and Lipaphis erysimi (Aphididae). —NZJ. Sci, 21:389-392.

Machev, N. R & Schraudolf, H. 1978. Thiocyanate as predeces-sor of asparagine in Sinapis alba L, - Plant Physioi. (Bul-aaria)4: 26-33.

Machiin, S., Mitchell-Olds, T. & Bradley, D, 1993. Sequence ofa Brassica campestris myrosinase gene, - Plant Physiol.102: 1359-1360.

MacLeod, A. J. & Rossiter, J. T. 1985, The occurrence of activ-ity of epithiospecifier protein in some Cruciferae seeds, -Phytochemistry 24: 1895-1898.

- & Rossiter, J, T. 1987. Degradation of 2-hydroxybut-3-enyi-glucosinolate (progoitrin). -Phytochemistr) 26: 669-673,

Matile, Ph, 1980. "Die Senfolbombe": Zur Kompanimentierungdes Myrosinasesystems, - Biochem. Physiol. Pflanz, 175:722-731,

Nagashima, Z, & Uchiyama, M. 1959, Possibility that mwosi-nase is a single enzyme and mechanism of decomposition ofmustard oil glucoside by myrosinase. - Bull, Agric, Chem.Soc. Jpn. 23: 555-556.

Oginsky, E, L., Stein, A, E, & Greer, M. A. 1965. Myrosmaseactivity in bacteria as demonstrated by the conversion ofprogoitriT! to aoitrin. - Soc, Exp. Biol, Med, Proc. 119: 360-3M.

Ohtsuru, M. & Hata, T. 1972. Molecular properties of multipleforms of plant myrosinase, - Agric, Biol, Chem. 36: 2495-2,503,

- & Hata, T, 1973, Studies on the activation mechanism of themyrosinase by L-ascorbic acid. - Agric, Biol, Chem. 37:1971-1972.

- & Kawatani, H, 1979. Studies of the myrosinase from Wasa-bia japonica: Purification and some properties of Wasabimvrosinase, - Agric. Bioi. Chem. 43: 2249-2255.

- , Tsuruo, I. &. Hata, T. i973. The production and stability ofintraceilular myrosinase from Aspergillus niger. - Agric.Biol. Chem, 37:967-971.

Peche, K. 1913, Microchemical detection of myrosin. - J.Chem, Soc. 106: 79-80.

Physio!, Plam. 97, t996 207

Petroski, R, J, & Kwoiek, W. F 1985. Interactions cf a fungalthioglucoside glucohydrolase and cruciferous plant epith-iospecifier protein to form 1-cyanoepithioalkanes, imphca-tions of an allosteric mechanism, - Phytochemistry 24: 213-216,

- & Tookey, H, L. 1982, Interactions of thioglucoside gluco-hydrolase and epithiospecifier protein of cruciferous plantsto form 1-cyanoepithioalkanes, — Phylochemistry 21: 1903—1905.

Pihakaski, K, & Iversen, T,-H, 1976, Myrosinase in Brassi-caceae, I, Localization of myrosinase in cell fractions ofroots of 5jn<jpis alba L, - 3. Exp. Bot. 27: 242-258,

Reese, E, T,, Clapp, R, C. & Mandels, M. 1958, A thioglucosi-dase in fiingi, - Arch. Biochem. Biophys. 75: 228-242,

Rest, J, A, & Vaughan, J. G, 1972, The development of proteinand oil bodies in the seed of Sinapis alba L, - Pianta 105:245-262,

Rodman, J, E, 1991, A taxonomic analysis of glucosinolate-pro-ducing plants. Part I: Phenetics, - Syst, Bot, 16: 598-618,

Schnug, E, 1990, Glucosinolates - fundamental, environmentaland agricultural aspects. - In Sulfur Nutrition and Sulfur As-similation in Higher Plants (H, Rennenberg, C, Brunold, L,J. de Kok and I, Stulen, eds), pp. 97-106. SPB AcademicPublishing, Hague, ISBN 90-5103-038-X.

- , Haneklaus, S,, Borchers, A, & Polle, A, 1995, Relationsbetween sulphur supply and glutathione and ascorbate con-centrations in Brassica napus. - Z. Pflanzenemahr.Bodenkd, 158: 67-69,

Searle, L, M,, Chamberlain, K,, Rausch, T, & Butcher, D, N,1982. The conversion of 3-indolylmethylglucosinolate to 3-indolylacetonitrile by myrosinase, and its relevance to theclute-oot disease of the Cruciferae. - J, Exp. Bot, 33: 935-942,

Spatzier, W. 1893. Uber das Auftreten und die physioiogischeBedeutung des Myrosins in der Pflanze, - Jahrb, Wiss. Bot.25: 39-79.

Springett, M. B. & Adams, J, B, 1989. Properties of brusselssprouts thioglucosidase. - Food Chem. 33: 173-186.

Sukhija, P S., Loomba, A., Ahuja, K, L, & Munshi, S. K. 1985.

Glucosinolate and liquid content in developing and germi-nating cruciferous seeds, — Plant Sci, 40: 1—6,

Tani, N., Ohtsuru, M, & Hata, T, 1974, Isolation of myrosinaseproducing microorganism, - Agric, Biol, Chem, 38: 1617-1622,

Thangstad, O. P, Iversen, T,-H,, Slupphaug, G. & Bones, A,1990, Immunocytochemical localization of myrosinase inBrassica napus L, - Pianta 180: 245-248,

- , Evjen, K. & Bones, A. 1991. Immunogold-EM localizationof myrosinase in Brassicaceae. - Protoplasma 161: 85-93,

- , Winge, P., Husebye, H. & Bones, A. 1993, The thiogluco-side glucohydrolase (myrosinase) gene family in Brassi-caceae, - Plant Mol. Bioi. 23: 511-524.

Tookey, H, L. 1973, Crambe thioglucoside glucohydrolase (EC3,2.3.1): Separation of a protein required for epithiobutaneformation. -Can, J, Biochem, 51: 1654-1660.

Tsuruo, I, & Hata, T. 1968. Studies on myrosinase in mustardseeds. Part IV, Sugars and glucosides as competitive inhibi-tors, - Agric, Biol, Chem, 32: 1420-1424.

VanSteenhouse, J. L., Fettman, M. J. & Gould, D, H, 1991. Theeffect of glutathione depletion by buthione sulphoximine onl-cyano-3,4-epithiobutane toxicity, - Food Chem. Toxicol.29: 153-157,

Wallig, M, A., Gould, D, H,, Fettman, M, J. & Willhite, C. C,1988. Comparative toxicities of the naturally occurring ni-trile l-cyano-3,4-epithiobutane and the synthetic nitrile n-valeronitrile in rats: Differences in target organs, metabo-lism and toxic mechanisms, - Food Chem. Toxicol. 26: 149-157,

Xue, J,, Lenman, M., Falk, A, & Rask, L, 1992. The glucosino-late-degrading enzyme myrosinase in Brassicaceae is en-coded by a gene family, - Plant Mol, Biol, 18: 387-398,

- , Philgren, U. & Rask, L. 1993. Temporal, cell-specific, andtissue-preferential expression of myrosinase genes duringembryo and seedling development in Sinapis alba. - Pianta191:95-101,

- , Jergensen, M., Philgren, U. & Rask, L. 1995. The myrosi-nase gene family in Arabidopsis ihaliana: gene organisation,expression and evolution. - Plant Mol. Biol. 27: 911-922.

Edited by L. J, Feldman

208 Ptiysiol. Plant. 97, 1996

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