PHYSIOLOGIA PLANTARUM 89: 811-816. 1993Printed in Denmark - all rights resened

Cops right (£) Physiologic! Planlarum 1993ISSN 0031-9317

Purification and characterization of a nitrilase from Brassica napus

Lara A. Bestwick, Line M. Gr0nning, David C. James, Atle Bones and John T. Rossiter

Bestwick, L. A., Gr0nning, L. M., James, D.C., Bones, A. and Rossiter, J.T. 1993.Purification and characterization of a nitrilase from Brassica napus. - Physiol. Plant.89: 811-816.

In germinating seedlings of Brassica napus glucosinolate levels decrease and arepotentially degraded to nitriles by a myrosinase. Little is known about the metabolismof glucosinolate aglycone products and the objective of this work was to investigatenitrilase activity and carry out a purification of the enzyme from seedlings of fi. napus.A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid waspurified to apparent homogeneity from seedlings of B. napus. The protein has amolecular mass of approximately 420 kDa made up of 38 kDa subunits. The pi of thenative protein was found to be 4.6. Under denaturing conditions on an isoelectricfocusing (IEF) gel a major and minor protein was observed with pi in the range of5.4-5.9, suggesting the presence of isoforms. Apart from the potential role of thenitrilase in indole-3-acetic acid (lAA) synthesis a developmental study with seedlingsindicates that the increase in activity observed may be linked to the in vivo degradationof glucosinolates.

Key words - Brassica napus, glucosinolate, nitriles, nitrilase, rapeseed. , .*

L. A. Bestwick, D. C. James and J. T. Rossiter (corresponding author), Dept of Biologi-cal Sciences, Wye College, Univ. of London, Ashford, Kent TN255AH, UK: LM.Gr0nning and A. Bones, UNIGEN-Centre for Molecular Biology, Dept of Botany,Univ. of Trondheim, Medisinsk Teknisk Senter, N-7005 Trondheim, Norway.

Introduction

The seeds of Brassica napus contain glucosinolateswhich are a potential source of nitrogen, sulphur andglucose nutrients. Glucosinolate degradation involves en-zymatic cleavage of the thioglucosidic linkage by myro-sinase ()S-thioglucosidase glucohydrolase, EC 3.2.3.1)yielding D-glucose and an unstable thiohydroximate-0-sulphate that spontaneously rearranges, resulting in theproduction of a sulphate ion and one of a wide range ofpossible reaction products. The products are generally athiocyanate, isothiocyanate or nitrile depending on suchfactors as substrate, pH or the availability of ferrous ions(Chew 1988). There is strong evidence that, in vivo,glucosinolates and their volatile degradation productshave a defensive role against pests and diseases. How-ever, it is also likely that glucosinolates may serve as anutrient source on seed germination. We have shown thata non-glycosylated form of myrosinase (myrosinase II)may be involved in aliphatic glucosinolate degradation

during early seedling growth (James and Rossiter 1991).The evidence for this comes from the de novo biosynthe-sis of myrosinase II in the cotyledons of B. napus to-gether with a decrease in aliphatic glucosinolates in thisorgan. It is likely that the in vivo degradation of glucosi-nolates gives rise to nitriles since hydrolysis to corre-sponding carboxylic acids would release nitrogen forbiosynthetic pathways.

Recent work (Bartling et al. 1992) has demonstratedthe presence of a nitrilase in Arabidopsis which canmetabolize indole-3-acetonitrile (IAN) to the plantgrowth hormone indole-3-acetic acid (IAA). In additionthis enzyme can metabolize a variety of nitriles to theircorresponding carboxylic acids (Bartling et al. 1992).The gene for this enzyme has been cloned and function-ally expressed in Escherichia coli. Earlier work with B.napus nitrilase (Rausch and Hilgenberg 1980) capable ofconverting IAN to IAA has been studied in tissues in-fected with clubroot disease (Rausch et al. 1981). How-ever, little information is available on the properties of

Received 24 May, 1993; revised 3 September, 1993

Physiol. Plant. 89. 1993 81

this enzyme. In order to understand the in vivo fate ofglucosinolates we have set out to purify and characterizethis enzyme. Unlike previous methods for assaying nitri-lase activity by measuring nitrogen or detecting IAA byan enzyme-linked immunosorbent assay (ELISA), a sim-ple assay based on the conversion of ['"^CNJ-phenyl-proprionitrile to ['^COOH]-phenylpropionic acid was em-ployed. Phenylpropionitrile is a potential in vivo catabo-lite of phenethylglucosinolate.

Abbreviations - DMSO, Dimethyl sulphoxide; IAA, indole-3-acetic acid; IAN, indole-3-acetonitril; IEF, isoelectric focusing;Mr, relative molecular mass; PMSF, phenylmethyl sulphonylfluoride.

Materials and methods

Plant material

Single-low oilseed rape seed {Brassica napus L. cv.Bienvenu) was sown in deep seed trays on 3 mm filterpaper moistened with tap water. The trays were coveredwith aluminium foil and seedlings were grown at 25°C indarkness in a controlled environment room.

Synthesis of ['''CN]-3-phenylpropionitrile

Phenethylbromide (100 mg, 0.54 mmol) was dissolved in300 JLl DMSO and 10 mg KCN (0.15 mmol) in 200 |xlDMSO added. The reaction was warmed to 60°C for 10min and 37 MBq of ['^Cj-KCN (1.85-2.22 TBq mol"') in200 |xl DMSO added. After 30 min cold KCN (30 mg,0.46 mmol) in 200 |xl DMSO was added to the mixtureand the reaction allowed to proceed for a further 30 min.The cooled solution was poured onto 6 ml water, ex-tracted with diethyl ether (3 x 1 ml) and the combinedorganic layers washed with 6 M HClaq, water, dried overMgS04, filtered and evaporated. The residue was distilledunder reduced pressure (Klihn-Roth apparatus) to givepure phenylpropionitrile (49.6 mg, 0.38 mmol, yield70.1%, radiochemical yield 44.6%, specific activity 43.7GBq mol-').

Nitrilase assay

['^CN]-Phenylpropionitrile (0.1 mM, 2.22 KBq, 43.7GBq mol-') was added to 0.05 M Tris buffer (pH 7.5)containing the enzyme (10-100 fxl) in a total volume of500 |JL1 and incubated at 35°C for 30 min. The reactionwas terminated by addition of 2 M NaOHa^ (100 JJLI) andtoluene (700 )JL1) added and the mixture vortexed for 20 s.After centrifugation (5 000 g for 2 min) 200 fxl of thetoluene extract was added to 3 ml scintillant (Ecolite,ICN) and remaining concentration of ['"'C]-phenylpropio-nitrile determined. Formation of the product ['^COOH]-3-phenylpropionic acid was confirmed by TLC on silicagel (Merck) using a hexaneiethyl acetate (1:1) solventsystem.

Purification of nitrilase

All chromatographic steps were carried out with a Waters650E Advanced Protein Purification System equippedwith a 600E system controller and Model 484 TunableAbsorbance Detector.

About 100 g of 4-day-old seedlings were blotted ontofilter paper and homogenized using an ice-cold pestle andmortar in 100 ml ice-cold 0.525 M Tris-HCl, 15 mM2-mercaptoethanol, 3 mM EDTA, 10 |xg ml"' leupeptin,0.1 mM PMSF, pH 7.5 and 10% (w/v) of polyvinylpoly-pyrrolidone.

The homogenate was squeezed through Miracloth(Calbiochem) and the filtrate was centrifuged at 100 000g. The supematant was desalted on a Sephadex-G25(5x12 cm) in 20 mM Tris-HCl (pH 7.5), 5 mM 2-mercaptoethanol, 1 mM EDTA and 0.04% sodium azide.Pre-swollen DEAE 'Fast Flow' Sepharose was obtainedfrom Pharmacia and Sephacryl S-300-HR from Sigma.The DEAE Sepharose anion exchange column (2.6 x 8cm) was equilibrated with 20 mM Tris-HCl, pH 7.5, 5mM mercaptoethanol, 1 mM EDTA and 0.04% sodiumazide (buffer A). Approximately 80 mg of desalted pro-tein was applied for each successive run. Unbound pro-tein was washed off with 100 ml of buffer A (2 ml min"')and bound proteins eluted with a 0-0.4 M NaCl gradientin buffer A, total volume 220 ml, followed by a 0.4-1.0M NaCl gradient, total volume 40 ml. Active fractionswere combined and concentrated by ultrafiltration (Ami-con DIAFLO membrane M^ cut-off 10000).

The concentrated active fractions were applied to aSephacryl S-300-HR column (2.6x60 cm) equilibratedwith 0.175 M Tris-HCl, pH 7.5, 5 mM mercaptoethanol, 1mM EDTA and 0.04% sodium azide. Proteins were elutedat a flow rate of 0.75 ml min"' and the active fractionscollected. Active fractions from the gel filtration stepwere combined and concentrated by ultrafiltration (Ami-con DIAFLO M, cut-off 10000) and desalted on Sepha-dex G25 equilibrated with buffer A.

Protein determinations

Protein concentration was estimated by the dye-bindingmethod of Bradford (1976) supplied as a kit by Bio-Rad.Bovine serum albumin was used as a standard.

Gel electrophoresis

Polypeptides were resolved in 13.5% (w/v) total acryl-amide vertical slabs according to the procedure ofLaemmli (1970) with Bio-Rad Mini Protean II electro-phoretic apparatus. Polypeptides were stained with0.25% (w/v) Coomassie Blue R-250 in 40% (v/v) metha-nol, 10% (v/v) acetic acid.

Isoelectric focusing experiments were carried out in0.75 mm x 10 cm polyacrylamide gels, T = 5%, C = 3.3%.The gels were prepared according to the procedure ofBollag and Edelstein (1991) using a pH gradient of 3-6.5.

812 Physiol. Plant. 89, 1993

Tab. 1. Purification of Brassica napus nitrilase from 4-day-old etiolated seedlings. The results are from a single experiment. Thepurification was repeated several times with similar results.

PurificationStep

Crude ]Desalted >DEAE-SepharoseSephacryl S-300-HR

, Protein' (mg)

14110114.62

Total activity(nmol min"')

10514691114473

Specific activity(nmol mg-' min"')

0.7514.676.4230

Yield' (%)

100^ 75.9

32.2

Purification

• = : - ' .; ' 1

^ . . . - ^ 5.2 ''15.7

The experiments were run at 0.5 h at 150 V followed by2.5 h at 200 V. Staining of the gel was carried out withCoomassie blue as described for the SDS-PAGE gel.

Carbohydrate analysis was carried out using Glyco-track^"^ (Oxford Glycosystems) coupled with enhancedchemiluminescence detection (Amersham).

pH optimization

Desalted crude extracts were assayed in three differentbuffer systems (Citric acid-phosphate buffer, pH 5.0-7.5;Tris-HCl buffer, pH 7.1-8.9; Glycine-NaOH buffer, pH8.5-10.6).

pi determination

The pi of the native protein was determined on a Mono Pcolumn using a pH gradient generated with Polybuffer7-4 (Pharmacia, Uppsala, Sweden).

Developmental study

One hundred seeds were used for each time-point andharvested on days 1-10 and nitrilase activity determined.These experiments were carried out in triplicate.

Temperature optimization

Desalted crude extracts were pre-incubated for 5 min andsubsequently assayed at 15, 25, 35, 45 and 55°C for 30min.

97-

6 6 -

43-

29-

20-

Fig. I. SDS-PAGE of the purification of B. napus nitrilasestained with Coomassie Blue R-250. M, molecular massmarkers; 1, crude extract, 30 |JLg; 2, diaylsed extract, 30 ^.g; 3,DEAE Sepharose fraction, 30 |xg; 4, Separcryl S300-HR frac-tion, 5 |xg. Molecular mass markers (Sigma) were phosphory-lase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin(45 kDa), bovine carbonic anhydrase (29 kDa), trypsin inhibitor(20.1 kDa).

Km determination

This was determined on crude dialysed, ion-exchangeand gel filtration fractions using substrate concentrationsfrom 0.04 mM to 0.2 mM and calculated using a Line-weaver-Burk plot.

ResultsPurification of nitrilase

The nitrilase was isolated from 4-day-old seedlingsgrown in darkness at 25°C. A developmental studyshowed that enzyme activity was high at this point andplant material was easily obtained.

The purification procedure involved two fractionationsteps (Tab. 1) involving desalting on Sephadex G25,chromatography on DEAE 'Fast Flow' Sepharose andHigh Resolution Sephacryl-300. The ion exchange andgel filtration stages provided 15.7- and 5.2-fold puri-fication, respectively, from desalted extract. The low totalactivity in the crude extract compared to the desaltedfraction is likely due to the presence of low molecularweight inhibitors of the enzyme. The purity of the finalpreparation was assessed by SDS-PAGE (Fig. 1) andshowed one main band to be present. Early preparativework showed the enzyme to be unstable. This instabilitywas shown to be temperature dependent with the enzymeafter 21 h losing ca 50% and ca 25% of its activity atroom temperature and 4°C, respectively. The addition ofprotease inhibitors had little effect on the stability of theenzyme and activity was best maintained by carrying outall procedures at <4°C. However, post-ion exchangepreparations were found to be relatively stable for a fewweeks at 4°C

Physical and kinetic properties

The nitrilase had an apparent molecular mass of 420 ± 10kDa (determined by gel filtration) while the analysis ofthe denatured protein by SDS-PAGE indicated that the Mr

Physiol. Plant. 89. 1993 813

- 20

5.1-

4.6-

3.6-

MFig. 2. Urea-PAG IEF of purified nitrilase stained with Coomas-sie Blue R-250. M, pi markers; 1, purified nitrilase, 5 [x.g. pimarkers (Sigma) were bovine carbonic anhydrase II (5.9), bo-vine carbonic anhydrase II (5.4), /3-lactoglobulin A (5.1), tryp-sin inhibitor (4.6) and amyloglucosidase (3.6).

10 20 30 40 50 60

Temperature (°C)

Fig.4. Temperature optimization of nitrilase activity of etiolatedB. napus seedlings. T"he assay was carried out in triplicate. Thebars represent standard error of the mean (SE).

of subunit polypeptide was 38 000 ±2 000. These datasuggest that the nitrilase exists as a multimer. The pi ofthe native protein was estimated to be 4.62 by isoelectricfocusing on a Pharmacia Mono P column. Under dena-turing conditions on a urea-IEF gel, a major and a minorband was observed with pi values in the range of 5.4-5.9indicating the presence of 2 isoforms (Fig. 2). The dif-ferences between native and denatured pi values arelikely to be due to changes in protein conformation. Thepurified nitrilase was analysed for the presence of carbo-hydrate after SDS-PAGE and negligible quantities werefound.

The Km of the nitrilase with phenylpropionitrile wasfound to be 0.273, 0.111 and 0.043 mM for crude dia-lysed, post-ion exchange and gel filtration fractions, re-spectively. The decreasing K,,, during purification demon-strates the removal of interfering agents allowing in-creased affinity of the nitrilase for the substrate.

The partially purified enzyme (ion exchange fraction)exhibited activity in a fairly narrow pH range with opti-mal activity at pH 9.0 (Fig. 3).

The 50 mM Tris-HCl buffer gave slightly higher ac-tivity than the 50 mM glycine-NaOH buffer. This value issimilar to that obtained (pH 9.2) for the Klebsiella ozae-nae nitrilase (Stalker et al. 1988).

The temperature optimum is around 35°C (Fig. 4) afterwhich there was a sharp decrease in nitrilase activity andagain is similar to the Klebsiella ozaenae nitrilase.

Developmental study

The developmental study showed a steady increase innitrilase activity (Fig. 5) to day 4 after which there was amarked decline. No nitrilase activity could be demon-strated in seed material.

4 5 6 7 8 9pH

Fig. 3. pH optimization of nitrilase activity in 100 mM citrate-phosphate (D), 50 mM Tris-HCl (<)) and 50 mM glycine-NaOH(O). The assays were carried out in duplicate. • '

i 450T 400

i» 350

I

I

I

250

200

150

100

50

0

1

-

-

-

1

1

/

1

1

/

/

1

1 1

T

/A

\

\

1 1

1

>

-

0 1 102 3 4 5 6 7 8 9

Days after sowingFig. 5. Total nitrilase activity in 100 etiolated B. napus seed-lings. Each point is the mean of three extractions which werecarried out in triplicate. Bars represent SE. '

814 Physiol. Plant. 89. 1993

Discussion

We have shown that a nitrilase present in developingseedlings of fi. napus can be purified in two stages and inhigh yield. This protein is not present in seed but israpidly synthesized over a period of about 4 days afterwhich activity markedly declines. This would indicatethat the protein is developmentally regulated during earlyseedling growth and that a 'coarse' metabolic control isoperating. The nitrilase is not glycosylated and maytherefore be susceptible to in vivo protease degradation(Faye et al. 1989). Additionally, in vitro instability of theprotein supports the notion that rapid turnover of theenzyme in the seedling may exist.

This protein is likely to be similar to the nitrilaserecently cloned from Arabidopsis by immunoscreening acDNA library with antibodies raised to pure plasmamembranes (Bartling et al. 1992). These authors reportmolecular masses of 37.5 and 38.2 kDa polypeptidesconsisting of 340 and 346 amino acids, respectively. Theamino acid sequence of the Arabidopsis nitrilase wasfound to resemble the Klebsiella ozaenae bromoxynilnitrilase (Stalker et al. 1988) and similar homologies existwith a more recently cloned nitrilase from Alcciligenesfaecalis (Kobayashi et al. 1993). The results of our SDS-PAGE indicate a similar molecular mass (38 ±2 kDa) tothat of the Arabidopsis and bacterial nitrilases. However,our work demonstrates the multimeric nature of the en-zyme in its native form. The K. determination (0.043mM) of the purified B. napus nitrilase would indicate thatit has a high affinity for phenylpropionitrile.

It is generally accepted that the plant hormone IAA issynthesised from tryptophan. However, several routesfrom tryptophan to IAA are possible via tryptamine (e.g.Phelps and Sequeira 1967) indole-3-pyruvate (e.g. Gib-son et al. 1972) and indole-3-acetaldoxime (Helminger etal. 1987, Ludwig-Muller and Hilgenberg 1988). In addi-tion the Arabidopsis nitrilase (Bartling et al. 1992) hasbeen shown to metabolize IAN to IAA. With our enzymethere would appear to be at least two isoforms present inthe purified preparation which might suggest that eachisoform may have different substrate specificities to-gether with different mechanisms of regulation.

In the Brassicaceae it is possible that IAN derived fromindoleglucosinolate may be a precursor for IAA via hy-drolysis by a nitrilase (Rausch and Hilgenberg 1980).This may be one of the roles for the nitrilase from B.napus. However, there is also some evidence, albeit cir-cumstantial, that the nitrilase we have purified maybeinvolved in the in vivo degradation of glucosinolates.Degradation of glucosinolates in vivo is likely to releaseglucose and sulphate for use in biosynthetic pathways.However, the in vivo fate of the aglycone products isobscure but it is known from autolytic work that glucosi-nolates can give rise to nitriles in both seed and maturetissues. The question then arises as to how the aglyconeproducts are catabolized in vivo. We believe a salvagepathway exists in B. napus for the alkenyl and hydroxyal-

kenyl glucosinolates in developing seedlings and forother glucosinolates throughout the life cycle of the plant.The evidence for this comes from recent observationsexamining glucosinolate concentrations and myrosinaseisoforms in B. napus. In single-low varieties, the seedlingcontent of aliphatic glucosinolates diminishes markedlyover a period of 7 days (Uppstrom 1983, McGregor1988). The majority of aliphatic glucosinolate loss occursin the cotyledons. In addition. Cole (1978, 1980) reporteda decrease in aliphatic glucosinolate content of turnip,cabbage, fodder rape, cauliflower and radish seedlings,over the first few days of seedling growth.

Furthermore, it has been shown for B. napus and othercmcifers that while total glucosinolate levels drop duringearly seedling growth, endogenous ascorbate (an activa-tor of myrosinase) concentration increases (Sukhija et al.1985). We have suggested that in B. napus myrosinase IImay be involved in this process of aliphatic glucosinolatedegradation during early seedling growth. The exclusivecotyledonary location of myrosinase II supports this hy-pothesis. Aliphatic glucosinolate degradation in the seed-ling, especially in the storage organs, the cotyledons, canperhaps be considered to be a mobilization of 'reserve'compounds by a specific mobilizing enzyme, myrosinaseII, synthesized de novo. The role of the nitrilase would beto release nitrogen from the aliphatic aglycones for use inother biosynthetic pathways. However, the potential rolesof nitrilase in B. napus is still open to debate. The fate ofnitriles and carboxylic acids derived from the glucosino-lates in B. napus is currently under investigation.

Acknowledgements - J.T. Rossiter and A. Bones wish to thankthe Norwegian British Council and NAVF for a travel grant.

References

Bartling, D., Seedorf, M., Mithofer, A. & Weiler, E.W. 1992.Cloning and expression of an Arabidopsis nitrilase whichcan convert indole-3-acetonitrile to the plant hormone, in-dole-3-acetic acid. - Eur. J. Biochem. 205: 417-424.

Bollag, D. M. & Edelstein, S.J. 1991. Isoelectric focusing andtwo dimensional gel electrophoresis. - In Protein Methods(D. M. Bollag and S.J. Edelstein, eds), pp. 161-180. Wiley-Liss, New York, NY. ISBN 0-471-56871-6.

Bradford, M.M. 1976. A rapid and sensitive method for thequantitation of microgram quantites of protein utilizing theprinciple of protein-dye binding. - Anal. Biochem. 72: 248-254.

Chew, F. S. 1988. Biological effects of glucosinolates. - InBiologically Active Natural Products - Potential Use inAgriculture (H. G. Cutler, ed.), pp. 155-181. AmericanChemical Society, Washington DC. ISBN 0-8412-1556-1.

Cole, R. A. 1978. Epithiospecifier protein in turnip and changesin products of autolysis during ontogeny. - Phytochemistry17: 1563-1565.

- 1980. Volatile components produced during ontogeny ofsome cultivated species. - J. Sci. Food Agric. 31: 549-557.

Faye, F, Johnson, K.D., Sturm, A. & Chrispeels, M.J. 1989.Structure, biosynthesis and function of asparagine-linkedglycans on plant glycoproteins. - Physiol. Plant. 75: 309-314.

Gibson, R. A., Schheider, E. A. & Wightman, F. 1972. Bio-synthesis and metabolism of indol-3-yl-acetic acid. II. In

53 Physiol. Plant. 89, 1993 815

vivo experiments with labelled precursors of IAA, in tomatoand barley shoots. - J. Exp. Bot. 23: 381-399.

Helminger, J., Rausch, T. & Hilgenberg, W. 1987. A solubleprotein factor from Chinese cabbage converts indole-3-acetaldehydeoxime to IAA. - Phytochemistry 26: 615-618.

James, D.C. & Rossiter, J.T. 1991. Development and charac-teristics of myrosinase in Brassica napus during early seed-ling growth. - Physiol. Plant. 82: 163-170.

Kobayashi, M., Izui, H., Nagaswa, T. & Yamada, H. 1993.Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: Cloning of the Alcali-genes gene and site directed mutagenesis of cysteine re-sidues. - Proc. Natl. Acad. Sci. USA 90: 247-251.

Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. - Nature 227:680-685.

Ludwig-Muller, J. & Hilgenberg, W. 1988. A plasma membranebound enzyme oxidises L-tryptophan to indole-3-acetal-doxime. - Physiol. Plant. 74: 240-250.

McGregor, D.I. 1988. Glucosinolate content of developing

rapeseed (Brassica napus L. 'Midas') seedlings. - Can. J.Plant Sci. 68: 367-380.

Phelps, R. H. & Sequeira, L. 1967. Synthesis of indoleaceticacid via tryptamine by a cell free system from tobaccoterminal buds. - Plant Physiol. 42: 1161-1163.

Rausch, T. & Hilgenberg, W. 1980. Partial purification of nitri-lase from Chinese cabbage. - Phytochemistry 19: 747-750.

- , Butcher, D. N. & Hilgenberg, W. 1981. Nitrilase activity inclubroot diseased plants. - Physiol. Plant. 52: 467^70.

Stalker, D. M., Malyj, L. D. & McBride, K. E. 1988. Purificationand properties of a nitrilase specific for the herbicide bro-moxynil and corresponding nucleotide sequence analysis ofthe bxn gene. - J. Biol. Chem. 263: 6310-6314.

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.

Uppstrom, B. 1983. Glucosinolate pattern in different growthstages of high and low glucosinolate varieties of Brassicanapus. - Sver. Utsadesforen. Tidskr. 93: 331-336.

Edited by C. H. Bomman

r

816 Physiol. Plant. 89, 1993