Proc. Nati. Acad. Sci. USA Vol. 91, pp. 2166-2170, March 1994 Biochemistry Redirection of tryptophan leads to production of low indole glucosinolate canola (Brtwc na/e SUPA CHAVADEJ*, NORMAND BRISSONt, JEREMY N. MCNEIL*, AND VINCENZO DE LUCA* *Institut de Recherche en Biologic V6gtale, Department of Biolcal Sciences, Universit6 de Montr&al, 4101 Sherbrooke Sreet East, Montrtal, QuEbec, Cafada H1X 2B2; tDepartment of Biochemistry, Universit6 de MontrEal, MontrEal, QuEbec, Canada H3C 3J7; and *Department of Biology, UniversitE Laval, QuEbec, Canada G1K7P4 Communicated by E. E. Conn, December 6, 1993 (receivedfor review October 4, 1993)

ABSTRACT Cruferous plants are known to produce One strategy to increase our understanding of metabolic over a hundred dfferent musard oil glycosides, which are networks that regulate the shikimate pathway is to redirect derived fom m oe, phenyllanine, or t p n. n the flow ofa key metabolite and to analyze the consequences il-producing crops Aike Brasuia napus (canola), the presence on the rest of the pathway. Glucosinolates are secondary of In seed protein meals has decred products typically found in the family Cruciferae (5) that are meal atbbty and has limited their value as animalfeed. We derived from the amino acids methionine, phenylalanine, and have tr f canola plants with a gene that en tryptophan. The biological function of these sulfur-con- tryptophan decarbo e (DC) in an attempt to redirect taining compounds is not well understood, but defensive tryptophan Into pt e rather than into indole g roles against pathogenic bacteria, fungi, and insect herbi- lates. c plants that expssed this decarboylse vores have been proposed (6). The availability ofplants with activity accumulated typtaI while coyd lower altered levels of specific glucosinolates could be used to levels of try t -derived iole gin inltes were pro- investigate the biological function of these metabolites. In duced in all at parts c ared with trnsr con- this report, we demonstrate that transformation of Brassica trol. Of p r , the idole gl iate co- napus cv. Westar with a tryptophan decarboxylase (TDC) tent ofmature eds fm t plants as only 3% ofthat gene (7), isolated from the medicinal plant Catharanthus found In O H- seeds. 11es reults d a ehow roseus, results in plants that divert the key metabolite, the creatioofa c eabolic sinks could divert me tryptophan, into tryptamine rather than into indole gluco- flow and be used to remove these undesirable indole ginco- sinolates (Fig. 1). snotes, thereby increasing the value of the oilseed meals, which are produced after extraction of oil from the seed. MATERIALS AND METHODS Plants produce a rich diversity of secondary compounds, which do not seem necessary forbasic metabolism but appear Transformation of Canola. A single cDNA encoding the to contribute to their environmental fitness and adaptability. enzyme TDC, previously cloned from the medicinal plant C. From a human perspective, secondary metabolites are re- roseus (7), was placed under transcriptional control of the sponsible for the flavor, aroma, and color offoods, and they cauliflower mosaic virus 35S promoter. The plasmid vector are sources of numerous medicinal, toxic, and industrial was constructed by replacement of the P-glucuronidase chemicals. These important commercial and cultural uses of (GUS) gene between the Xba I and Sst I sites in the plant secondary metabolites have intensive cauliflower mosaic virus 35S Nos terminator cassette of encouraged stud- pBI121 (Clontech) with a 2-kb Xba I/Xho I fragment from ies on the regulation and control of biosynthetic pathways pBSKS+ (Stratagene) containing the full-length TDC cDNA and, more recently, made them important targets for meta- clone pTDC5 (7). The Xho I site of the fragment was linked bolic engineering. to the Sst I site by using the oligonucleotide adapter TC- Bailey (1) has defined metabolic engineering as "the im- GAGGAGCT. The construct was mobilized into the dis- provement of cellular activities by manipulation of enzy- armed Agrobacterium tumefaciens strain LBA 4404 by the matic, transport, and regulatory functions ofthe cell with the triparental mating procedure (8) and used to transform canola use of recombinant DNA technology." However, efforts to (B. napus cv. Westar) plants (9). Canola plants were also alter metabolic pathways in plants have produced unpredict- transformed with the pBI121 plasmid vector containing the able results (2) because of our poor understanding of meta- GUS gene as control. The seeds ofregenerated canola plants bolic networks (3), which take into account control architec- that expressed TDC or GUS activity were collected and used tures at key branch points and pathways as a whole rather for subsequent experiments. than individual reactions. For example, the shikimate path- Selecdon of myc-Restant Seding. Seeds of non- way with its many metabolic branches has been studied transformed control and of transgenic canola plants were extensively in microorganisms and more recently in plants surface sterilized in 70% ethanol for 2 min and in 3% sodium (4). This pathway is responsible for biosynthesis of the hypochlorite for 20 min. Seeds were washed in sterile dis- aromatic amino acids phenylalanine, tyrosine, and tryp- tilled water three times and were allowed to imbibe water for tophan as well as for biosynthesis of an endless variety of 3 h. Imbibed transgenic seeds were placed on filter paper phenolic compounds and alkaloids. However, it is poorly (Whatmann 3 MM) in Magenta vessels containing 8 ml of0.2 understood how metabolic precursors are distributed among MS salts (20%6 full-strength Murashige-Skoog salts) and 0.25 the many branch points known to exist. mg ofkanamycin per liter (Sigma), whereas control nontrans- formed seeds were grown in the absence ofkanamycin. Seeds The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GUS, f-glucuronidase; TDC, tryptophan decarbox- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ylase. 2166 Downloaded by guest on September 26, 2021 Biochemistry: Chavadej et al. Proc. Natl. Acad. Sci. USA 91 (1994) 2167

OH SGlucose indole glucosinolates, which are derived from methionine and tryptophan, respectively. Eighty-five putative transgenic plants, selected on kanamycin-containing medium, were al- NOSO3- lowed to flower and to set seed. Further Northern blot, H Western blot, enzymatic, and chemical studies revealed that I 11 plants actually expressed TDC activity (data not shown). 0.* COOH Expression of TDC Activity in Different Transgenic Canola Plants. Seven independent TDC-expressing transgenic N canola lines were studied (Fig. 2) and were shown to accu- H Tryptophan mulate tryptamine (Fig. 2A), the immediate decarboxylation product of the TDC reaction. The tryptamine that accumu- Decarboxylase lated in each transgenic plant was correlated with both TDC-specific activity (Fig. 2B) and relative TDC mRNA NH2 levels (Fig. 2C). In comparison, nontransformed control H plants and those transformed with the GUS gene accumu- lated neithertryptamine nor TDC mRNA (Fig. 2A and C) and FIG. 1. Transformation of canola with the TDC gene is expected expressed no TDC activity (Fig. 2B). The GUS controls also to redirect tryptophan into tryptamine rather than into the indole showed that the results obtained with TDC-expressing plants glucosinolates normally occurring in nontransformed plants. are not an artefact of transformation. The major indole glucosinolate found in canola leaves is were germinated at 21'C ± 20C in a 16-h light/8-h dark indol-3-ylmethylglucosinolate (structure I) (Fig. 3E) repre- photoperiod at a light intensity of 50 AE m-2-s-'. After 1 senting 90% of the total. Young fully expanded canola leaves week, seedlings were scored for resistance (expanded green from nontransformed or from GUS-transformed plants ac- cotyledons, elongated axis, and presence of roots) or sensi- cumulated between 98 and 122 nmol of indol-3-ylmethylglu- tivity (yellow embryo, axis not elongated) to kanamycin. cosinolate (structure I) per g fresh weight, whereas those Control nontransformed and resistant seedlings were trans- from TDC-transformed lines accumulated only 20-38 nmol of ferred to the greenhouse where they were grown under indol-3-ylmethylglucosinolate (structure I) per g fresh weight controlled conditions at a photosynthetic photon flux density Leaves from nontransformed or of 200 ,tE-m-2-s-1 and a light regime of 16 h oflight (210C) and (Fig. 3D). from GUS- 8 h was transformed control plants produce no tryptamine, whereas of dark (15'C). The soil composed of2 parts Pro-Mix those (pH 5.5-6), 2 parts topsoil, and 1 part Perlite. Plants were from TDC-transformed lines accumulated between 15 watered twice a day with tap water. and 115 nmol of tryptamine per g fresh weight (Fig. 2A). Analysis of Transgenic Plants. Plant materials were col- These results suggest that the tryptophan pool destined for lected, divided into 4 parts of 1 g fresh weight each, and the biosynthesis of indole glucosinolates is effectively redi- frozen in liquid N2 for analysis of TDC enzyme activity (10), rected and quantitatively converted into tryptamine in trans- TDC RNA levels, and tryptamine and glucosinolate content. genic lines expressing TDC activity (Fig. 2 A and D), whereas Tryptamine was extracted and analyzed as described (11). the levels ofallyl glucosinolates remained unaltered (data not Total RNA was extracted, processed for slot blot analysis (30 shown). ug of total RNA per slot), and hybridized with 32P-labeled Canola seeds contain two major indole glucosinolates, TDC probe (1600-bp EcoRI fragment); hybridizing RNA was indol-3-ylmethylglucosinolate (structure I) and 4-hydroxyin- detected by autoradiography as described by Maniatis et al. dol-3-ylmethylglucosinolate (structure II), in a 1:4 ratio, (12). Relative amounts of RNA hybridizing to the labeled respectively. Seeds from nontransformed or from GUS- probe were quantified by scanning laser densitometry. transformed canola plants accumulate between 6 and 6.5 Glucosinolate Analysis. Frozen plant materials were pul- ,umol of indole glucosinolate per g fresh weight, whereas verized in liquid N2 and extracted with boiling 100% methanol those from TDC-transformed lines accumulate between 0.2 and reextracted with boiling 70% methanol (13, 14). After and 3.6 ,umol ofindole glucosinolate perg fresh weight. Seeds removal of the volatile solvent in vacuo, the samples were of the transgenic line St 004, which has the lowest TDC stored at -20°C until required. Canola seed samples (30 activity, accumulate 50% of the indole glucosinolates of seeds; =100 mg) were ground and extracted twice with control plants. Seeds of lines St 053 and St 062, with the boiling 70%o methanol (15, 16) and stored at -20°C. Before highest TDC activities, accumulate only 3% of the indole analysis, extracts were thawed, applied to a microcolumn of glucosinolates found in control plants. In contrast, the allyl DEAE-Sephadex A25, treated with the enzyme sulfatase glucosinolates in seeds from all transgenic lines remained type H1 (Sigma), and eluted. These desulfated samples and essentially unaltered (Fig. 2F). Analysis of mature seeds standard desulfoglucosinolates were analyzed by HPLC as produced from each transgenic line demonstrated conclu- described by Quinsac and Ribailler (16). sively that redirection of tryptophan results in seeds contain- Preparation of Desulfoglucosinolate Standards. Allyl de- ing much lower levels of indole glucosinolates (Fig. 2E). sulfoglucosinolate and benzyl desulfoglucosinolate were Tissue-Specific Expression of TDC in Transgenic Line St prepared by enzymatic desulfation of commercial allyl 062. More detailed analyses of transgenic line St 062 clearly glucosinolate (Sigma) and benzyl glucosinolate (Merck), showed the relationship between TDC activity and the lower respectively. Desulfo-2-hydroxybut-3-enylglucosinolate, indole glucosinolate levels accumulating in different plant desulfo-2-hydroxypent-4-enylglucosinolate, desulfo-4- parts (Fig. 3). Protein extracts from 7-day-old seedlings, hydroxyindole-3-ylmethylglucosinolate, and desulfoindol- young leaves ofpreflowering plants, and fully opened flowers 3-ylmethylglucosinolate were isolated by semipreparative exhibited 6-fold higher levels of TDC activity than wild-type high-performance liquid chromatography of a desulfoglu- control plants, while extracts from 14-day-old roots, leaves of cosinolate mixture obtained by desulfating a large extract of postflowering plants, and green developing seeds contained canola seeds. TDC activities 3-fold higher than background values (Fig. 3D). With the exception of mature seeds in which few active enzymes are found, the TDC-specific activity of each plant RESULTS organ correlates with the quantity of tryptamine accumulated Introduction of the TDC Gene into B. napus cv. Westar. The (Fig. 3C) and with the marked decline of indole glucosinolate canola cultivar Westar produces both allyl glucosinolates and levels (Fig. 3A). This variable expression of TDC in different Downloaded by guest on September 26, 2021 2168 Biochemistry: Chavadej et al. Proc. Natl. Acad. Sci. USA 91 (1994)

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FIG. 2. Relationship between the level of TDC activity and accumulation of tryptamine and indole glucosinolates in different transgenic canola lines. Content oftryptamine (A), TDC activities (B), TDC mRNA (C), and indole glucosinolates (D) in young fully expanded leaves (solid bars) ofdifferent transgenic canola plants compared to wild-type (WT) plants and those transformed with the Escherichia coli GUS genes (GUS1 and GUS2). Plants were 5 weeks old and had not yet flowered. The content of seed (hatched bars) indole glucosinolates (E) and allyl glucosinolates (F) in transgenic canola plants is also shown compared to the same controls. Each measurement (mean ± SD) represents results from three separate plants for each control and each transgenic line. Background activities observed in controls (B) are artefacts of the assay procedure and do not represent TDC activity.

tissues reflects the activities normally observed with genes 2-hydroxybut-3-enylglucosinolate (structure VI) in a 1:10 placed behind the cauliflower mosaic virus 35S promoter ratio, which reflects reduced synthesis of indol-3-ylmethyl- (17). glucosinolate (structure I). Mature seeds of line St 062 The accumulation of total indole glucosinolates in mature accumulated greatly reduced levels of 4-hydroxyindol-3- seeds of line St 062 was 0.2 ± 0.045 pumol per g fresh weight ylmethylglucosinolate (structure II) and indol-3-ylmethylglu- of seed, compared with 6 ± 1 timol per g fresh weight for cosinolate (structure I), whereas the chain-extended deriva- untransformed seed (Fig. 3A). In contrast, seeds ofline St 062 tive of 2-hydroxybut-3-enylglucosinolate (structure VI), accumulated similar levels of allyl glucosinolates (16 ± 2 2-hydroxy-4-enylglucosinolate (structure VIII), almost dou- jumol per g fresh weight of seed) as untransformed seeds (13 bled in level. The ratio of 2-hydroxybut-3-enylglucosinolate ± 3 pAmol per g fresh weight of seed) (Fig. 3B). Apparently the (structure VI) to 2-hydroxy-4-enylglucosinolate (structure decreased availability of tryptophan for indole glucosinolate VIII) changed from 2:1 in control seeds to almost 1:1 in biosynthesis has no significant effect on production and transgenic seeds. Similar changes in ratio were also observed accumulation of the methionine-derived allyl glucosinolates. in each ofthe other transgenic lines studied (data not shown). The similar allyl glucosinolate content of transgenic and These results therefore suggest that even if the pathways for control roots, leaves, open flowers, and developing seedlings biosynthesis of allyl and indole glucosinolates are separate, (Fig. 3B) also supports the hypothesis that different biosyn- they may nevertheless be interdependent. thetic mechanisms may be involved for production of indole Effects of Low Indole Glucosinolates on Seedling Germina- and allyl glucosinolates (18-20). tion and Plant Growth. Quantitative estimates of seed quan- Canola plants accumulate various levels of allyl and indole tity per plant, germination rates, growth rates, time to glucosinolates (Fig. 3E) in different organs. The leaves flowering, and plant size were also determined for line St 062 accumulate predominantly indol-3-ylmethylglucosinolate and were compared to nontransformed canola plants. Iden- (structure I) and 2-hydroxybut-3-enylglucosinolate (structure tical results were obtained for transgenic and nontransformed VI) in a 1:3 ratio, whereas mature seeds accumulate pre- canola plants. The yield of seed per plant averaged between dominantly 4-hydroxyindol-3-ylmethylglucosinolate (struc- 4 and 5 g (1200-1500 seeds). The germination rates offreshly ture II), indol-3-ylmethylglucosinolate (structure I), 2-hy- harvested seeds were >90%, whereas after 1 year this droxybut-3-enylglucosinolate (structure VI), and 2-hydroxy- decreased to ==60%, while germination occurred within 2 4-enylglucosinolate (structure VIII) in a 3.8:1:6.7:3.2 ratio days of planting. Transgenic and nontransformed control (Table 1). In contrast, leaves of transgenic line St 062 plants displayed identical growth rates and they flowered accumulated indol-3-ylmethylglucosinolate (structure I) and between weeks 5 and 6 after planting. Full-grown plants were Downloaded by guest on September 26, 2021 Biochemistry: Chavadej et al. Proc. Natl. Acad. Sci. USA 91 (1994) 2169

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90 FIG. 3. Analysis of TDC-overexpress- 0 ing canola line St 062 (hatched bars) com- ao pared to nontransformed wild-type control 60 plants (solid bars). Levels of indole glu- cosinolates (A), allyl glucosinolates (B), 30 tryptamine (C), and TDC (D) in extracts of 7-day-old seedlings, 14-day-old roots, young leaves before flowering (preflower- ing leaves), open flowers, leaves after D H flowering (postflowering leaves), green de- 8 veloping seeds, and mature seeds. Struc- tures of glucosinolates found in B. napus cv. Westar are as follows: structure I, 6 COOH indol-3-ylmethylglucosinolate; structure ~~~ ~~Tryptophan II, 4-hydroxyindol-3-ylmethylglucosino- 0 4 late; structure III, N-methoxyindol-3- ylmethylglucosinolate; structure IV, a o. 4-methoxyindol-3-ylmethylglucosinolate 2 0'D II (indole glucosinolates) (E) and structure V, but-3-enylglucosinolate; structure VI, 2-hydroxybut-3-enylglucosinolate; struc- 0 ture VII, pent-4-enylglucosinolate; struc- ture VIII, 2-hydroxy-4-enylglucosinolate (allyl glucosinolates) (F). The structure of tryptamine is shown in G and the reaction catalyzed by TDC is shown in H. Results are given as means ± SD from three sep- arate plants for the wild-type control and for line St 062. Background activities ob- served in the wild-type control (D) are 00 artefacts ofthe assay procedure and do not represent TDC activity.

3- to 4-feet tall and they had a similar number of leaves, quality of the oil, and low levels of aliphatic glucosinolates, flowers, and flower pods. Field trials should be performed, greatly improving the quality of the protein meal (21). How- however, in order to obtain more definitive phenotype char- ever, the levels of indole glucosinolates remained unchanged acteristics. in these cultivars, which impeded further improvements in protein meal quality. Molecular approaches involving an- DISCUSSION tisense RNA have been suggested to remove all glucosino- lates from Brassica oil seeds, but the enzymology of gluco- Intensive plant breeding programs have successfully reduced sinolate biosynthesis is poorly understood and genes in- glucosinolate levels in Brassica oil seeds. Crosses made with volved in this biosynthetic pathway remain to be isolated. An the low glucosinolate Polish cultivar Boronowski produced alternative strategy reported here has demonstrated that "double low" commercial rapeseed cultivars, known as metabolic engineering of canola with the TDC gene reduces canola, which contained no erucic acid, greatly improving the whole plant and seed indole glucosinolates by competition for Downloaded by guest on September 26, 2021 2170 Biochemistry: Chavadej et al. Proc. Natl. Acad Sci. USA 91 (1994) Table 1. Accumulation of glucosinolates in transgenic line St 062 become mature. The lack of tryptamine in low indole glu- compared to control nontransformed canola cosinolate canola may thus alleviate the real possibility that one toxic metabolite has been replaced by another and Structure suggest that these low indole glucosinolate canola lines may Plant part I II VI VIII be economically useful. In addition, studies over three gen- Young erations of seeds have shown that the production of tryptamine in transgenic canola or the loss of indole gluco- Control 84 1 280 14 sinolates does not appear to affect growth and development St 062 24 0 250 29 of plants or seeds. Furthermore, neither seed size nor via- Mature bility is affected. Some previous studies have suggested that Control 1302 5010 8820 4200 indole glucosinolates are implicated in pest resistance (6), but St 062 0 210 7900 7512 our laboratory studies on the oviposition of diamondback Structures of the glucosinolates are shown in Fig. 3 E and F. moths found that transgenic low indole glucosinolate canola Results are expressed as nmol per g fresh weight. Young, young lines are not more susceptible than control plants to this pest leaves before flowering; Mature, mature seeds. (unpublished observations). However, additional laboratory and field studies will be required to examine the various the tryptophan precursor and redirecting it toward produc- agronomic traits of these transgenic plants in order to fully tion of tryptamine (Fig. 1). A similar strategy was recently understand the consequences of this approach and the eco- used to redirect the aminocyclopropane carboxylic acid nomic usefulness of this altered crop. intermediate away from ethylene biosynthesis (22). Ethylene levels were successfully decreased by >90% in transgenic This work was supported by the Natural Sciences and Engineering tomatoes expressing a bacterial aminocyclopropane carbox- Research Council of Canada (V.D.L., N.B., J.N.M.) and by the ylic acid deaminase gene. Fonds pour la Formation de Chercheurs et l'Aide A la Recherche du Other research has involved strategies to overproduce Quebec (V.D.L., N.B.). tryptamine in a tobacco model system (11). Transgenic plants 1. Bailey, J. E. (1991) Science 253, 1668-1675. expressing various constitutive levels ofTDC produced 4-45 2. De Luca, V. (1993) AgBiotech News Info. 6, 225-229. times greater TDC activity than controls, and tryptamine 3. Stephanopoulos, G. & Sinskey, A. J. (1993) Trends Biotechnol. 11, accumulated in transgenic plants to levels that were directly 392-3%. 4. Bentley, R. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 307-384. proportional to their TDC-specific activity. Although very 5. Fenwick, G. R., Heaney, R. K. & Mawson, R. (1989) in Toxicants similar results have been reported here with TDC-expressing ofPlant Origin, ed. Cheeke, P. R. (CRC, Boca Raton), Vol. 2, pp. canola (Fig. 1), these plants never expressed >9% of the 1-41. TDC-specific activity and only accumulated 2% of the 6. Fenwick, G. R., Heaney, R. K. & Mullin, W. K. (1983) CRC Crit. found in the most active transgenic tobacco line. Rev. Food Sci. Nutr. 18, 123-148. tryptamine 7. De Luca, V., Marineau, C. & Brisson, N. (1989) Proc. Natl. Acad. These results illustrate that unpredictable species-specific Sci. USA 86, 2582-2586. differences, which may be related to how the metabolic 8. Matzke, A. J. M. & Matzke, M. A. (1986) Plant Mol. Biol. 7, network is regulated, will also define the level ofproduct that 357-365. can be produced as a result of metabolic engineering. 9. Fry, J., Barnason, A. & Horsch, R. (1987) Plant Cell Rep. 6, 321-325. Previous studies (11) have also shown that transgenic 10. De Luca, V., Alvarez-Fernandez, J., Campbell, D. & Kurz, tobacco plants with the highest TDC activity accumulated >1 W. G. W. (1988) Plant Physiol. 86, 447-450. mg of tryptamine per g fresh weight in their leaves. 11. Songstad, D. D., De Luca, V., Brisson, N., Kurz, W. G. W. & Tryptamine, which has been suggested as a possible precur- Nessler, C. L. (1989) Plant Physiol. 94, 1410-1413. sor for auxin did not affect auxin levels in 12. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1989) Molecular biosynthesis (23), Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, tobacco plants (11), consistent with reports (24, 25) that Plainview, NY). auxins are derived from an intermediate involved in tryp- 13. Heaney, R. K. & Fenwick, G. R. (1980) J. Sci. Food Agric. 31, tophan biosynthesis rather than from tryptophan. Although 593-597. auxins were not measured in TDC-expressing canola, the 14. Bradshaw, J. E., Heaney, R. K., Fenwick, G. R. & McNaughton, I. H. (1983) J. Sci. Food Agric. 34, 571-580. apparently normal phenotype of the plants observed with 15. Thies, W. (1976) Fette-Siefen Anstrichmittel 78, 231-240. respect to germination rates, growth rates, time to flowering, 16. Quinsac, A. & Ribailler, D. J. (1991) Assoc. Off. Anal. Chem. 74, plant size, and appearance suggests that auxin levels were 932-939. also unchanged as a result of expression of the TDC gene. 17. Benfey, P. N., Ren, L. & Chua, N. H. (1989) EMBO J. 8, 2195- The normal growth and development of all seven trans- 2202. 18. Poulton, J. E. & Moeller, B. L. (1993) in Methods in Plant Bio- genic lines ofcanola suggest that only tryptophan destined to chemistry, ed. Lea, P. J. (Academic, New York), Vol. 9, pp. accumulate as indole glucosinolates is affected. If this is the 209-237. case, then the redirection of branch point substrates might 19. Ludwig-Muller, J. & Hilgenberg, W. (1988) Physiol. Plant 74, also be useful in decreasing levels of other undesirable 240-250. secondary metabolites, such as the allyl glucosinolates of 20. Bennet, R., Donald, A., Dawson, G., Hick, A. & Wallsgrove, R. (1993) Plant Physiol. 102, 1307-1312. canola and the toxic cyanogenic glycosides in major crops 21. Robbelen, G. & Thies, W. (1980) inBrassica Crops and WildAllies: such as sorghum and cassava. The potential applications of Biology and Breeding, eds. Tsunoda, S., Hinata, H. & Gomez- this strategy are numerous and offer alternative solutions to Campo, G. (Japan Sci. Soc. Press, Tokyo), pp. 285-300. problems often treated by antisense approaches. 22. Klee, H., Hayford, M. B., Kretzmer, K. A., Barry, G. F. & While mature canola seed from all transgenic lines con- Kishore, G. (1990) Plant Cell 3, 1187-1193. tained significantly lower levels of indole glucosinolates, no 23. Reinecke, D. M. & Bandurski, R. S. (1990) in Plant Hormones and their Role in Plant Growth and Development, ed. Davies, P. J. tryptamine or tryptamine derivatives could be detected in (Kluwer, Boston), pp. 24-42. these tissues (Fig. 2C). The occurrence of low levels of 24. Wright, A. D., Sampson, M. B., Neuffer, M. G., Michalczuk, L., tryptamine in green seeds (Fig. 2C) suggests that this product Slovin, J. P. & Cohen, J. D. (1991) Science 254, 998-1000. is either metabolized or insolubilized by the time seeds 25. Last, R. L. & Fink, G. R. (1988) Science 240, 305-310. Downloaded by guest on September 26, 2021