tryp biosynthsesis

The Plant Cell, Vol. 7, 921-934, July 1995 0 1995 American Society of Plant Physiologists

Tryptophan Biosynthesis and Molecular Genetics

Biochemical and

Elaine R. Radwanski and Robert L. Last‘ Boyce Thompson lnstitute for Plant Research and Section of Genetics and Development, Cornell University, Tower Road, Ithaca, New York 14853-1801

INTRODUCTION

Tryptophan is the one of the least abundant yet, in terms of energy, one of the most expensive to produce of the standard protein amino acids (Hrazdina and Jensen, 1992). The Iow leve1 of soluble tryptophan present in plants (1 to 15 pM) belies the importance of the amino acid and the pathway that produces it (Gilchrist and Kosuge, 1980). Animals, and some eubacteria, lack the ability to synthesize tryptophan and must obtain it from plant and microbial sources (Crawford, 1989; Bentley, 1990; Herrmann et al., 1992). This essential amino acid is required by animals for protein synthesis as well as for the production of other compounds, including the neurohormone serotonin and the vitamin nicotinic acid. The primary role of tryptophan biosynthesis in bacteria and fungi isto provide the tryptophan needed for protein synthesis. In contrast, plants algo use this pathway to provide precursors for synthesis of the hormone auxin, phytoalexins, glucosinolates, and both indole- and anthranilate-derived alkaloids. A few examples of these secondary products are shown in Figure 1. Tryptophan biosynthesis thus plays a direct role in regulating plant development, pathogen defense responses, and plant-insect interactions. Volatile indolics are also implicated in attracting pollinating animals. In addition to playing important ecological roles, a number of indole alkaloids have important pharmacological uses (see Kutchan, 1995, this issue). For example, the indole alkaloids vinblastine and vincristine are widely used anticancer drugs (Wiebe and Sipila, 1994).

The biosynthetic pathway for tryptophan, shown in Figure 2, exhibits a striking conservation in its biochemistry across kingdoms (identical substrates are converted into the same products by homologous enzymes at each step in prokaryotes and eukaryotes) that contrasts sharply with the rich variation in regulatory controls (Hütter et al., 1986; Crawford, 1989; Rose and Last, 1994). In the following discussion of tryptophan biosynthesis, we show that this biochemical conservation was fruitfully exploited in the isolation of the Arabidopsis structural gene cDNAs for each step in the pathway by heterologous hybridization screening and complementation of fscherichia coli mutations (Rose and Last, 1994).

To whom correspondence should be addressed.

Over the past four decades, the tryptophan biosynthetic pathway of bacteria and fungi has been characterized extensively (Hütter et al., 1986; Yanofsky and Crawford, 1987; Bentley, 1990). In contrast, little was known about the plant pathway until the late 1980s. This review highlights more recent results obtained largely from the application of molecular biological techniques to the characterization of the pathway in wild-type and mutant plants, particularly in Arabidopsis. We begin with a summary of genetic and molecular “tools” (e.g., mutants, mapped loci, cDNA and genomic clones, and antibodies) now available for use in investigations of this branch of the aromatic amino acid biosynthetic pathway. Next, we report specific information on each step in the plant tryptophan biosynthetic pathway. Discussion of enzyme subunit organization, regulation, and subcellular localization follows. Finally, we discuss recent results on tryptophan metabolism. Readers interested in indole alkaloid or chorismate biosynthesis are directed to the articles by Kutchan (1995, this issue) and Herrmann (1995, this issue).

TRYPTOPHAN BIOSYNTHESIS

Mutants and Molecular Probes

Compared with most other plant amino acid biosynthetic pathways, the availability of an extensive collection of mutants, clones, mapped loci, and antibodies is a unique feature of tryptophan biosynthesis. lnformation regarding the names of mutant loci, cloned genes, and genetic mapping data are sum- marized in Table I, and the biosynthetic pathway is illustrated in Figure 2. Of particular significance is the availability of Arabidopsis genetic mutants or antisense plants for nearly all steps in the pathway (with the exception of indole-3-glycerolphosphate synthase). Severa1 methods were employed to identify these mutants. Selection for resistance to the anthranilate analogs 5- or &methylanthranilate, illustrated in Figure 3, permitted the identification of recessive mutations in phosphoribosylanthranilate (PR-anthranilate) transferase (frpl ) and in the a (frp3) and (frp2) subunits of tryptophan synthase (Last and Fink,

922 The Plant Cell

H

H

N-O-SOi H

Figure 1. lndolic Compounds Produced by the Tryptophan Biosyn- thetic Pathway.

From top to bottom: tryptophan, indole-3-acetic acid (auxin), 3-thiazol- P’-yl-indole (camalexin, a phytoalexin), and indol-3ylmethylglucosino- late (an indole glucosinolate).

1988; Last et al., 1991; Li et al., 1995b; Zhao and Last, 1995; E.R. Radwanski and R.L. Last, unpublished data). The basis for this selection is that wild-type plants convert these anthranilate derivatives into toxic tryptophan analogs (5- and Cmethyl- tryptophan, respectively), whereas mutants blocked in this conversion should have reduced toxin biosynthesis. Two mechanisms are presumed to contribute to the cytotoxicity of these tryptophan analogs. First, they can cause tryptophan starvation by acting as false-feedback inhibitors of anthranilate synthase (see later discussion), the committing enzyme in the pathway (Moyed, 1960; Widholm, 1972). Second, they may be incorporated in place of tryptophan during protein synthesis, although published reports indicate that 5methyltryptophan is not incorporated into proteins in yeast (Schurch et al., 1974) or in plant (Cafharanfhus roseus) cell suspension cultures (Staheli et al., 1981).

Whereas anthranilate analog selection has yielded mutations throughout the pathway, severa1 other approaches targeted specific steps in tryptophan biosynthesis. For example, a collection of trpl mutants, which are defective in converting anthranilate to 5-PR-anthranilate and thus accumulate fluorescent anthranilate compounds, was obtained by screening for seedlings that fluoresce blue under UV light (Last and Fink, 1988; Rose et al., 1992; A.B. Rose and R.L. Last, unpublished results). Suppression of the blue fluorescent phenotype conferred by the trpl-100 mutation was then used to identify loss-of-function trp4 mutants, which are defective in one member of the triplicate gene family for the anthranilate synthase p subunit and accumulate reduced

concentrations of anthranilate mmpounds (Niyogi et al., 1993). Tryptophan synthase mutants were targeted specifically by selection for resistance to 5-fluoroindole, which is presumably converted to the toxic tryptophan analog 5-fluorotryptophan by the p subunit of tryptophan synthase (Barczak et al., 1995). Finally, selection for resistance to a-methyltryptophan led to the identification of a monogenic semidominant tryptophan overproducer mutant containing an anthranilate synthase with reduced sensitivity to inhibition by tryptophan (Kreps and Town, 1992).

As a mnplement to the mutant analysis, molecular cloning has permitted the analysis of clones encoding all of the enzymes of the Arabidopsis tryptophan biosynthetic pathway (Berlyn et al., 1989; Elledge et al., 1991; Last et al., 1991; Niyogi and Fink, 1992; Rose et al., 1992; Niyogi et al., 1993; Li et al., 1995a, 1995b; Radwanski et al., 1995) and cDNAs for the maize tryptophan synthase B (Wright et al., 1992). The availability

p-minobenzoate

isochorismate

C h o r i s m a t e H o g C”

O- Íi- CWH

amtl=ASAI ? 4 trp4= ASBl

Anthranilate Synthase (1)

Phosphoribosylanthranilate Synthase (2)

Anthranilate

trpl = PATI

4 ~ ~ ~ ~ ~ ~ h o ~ i b o s y l ~ t h r ~ i l a t e Isomerase (3)

w-CH=C- CH-CH-CHX)PO~HZ

Indole-3-glycerolphosphate Synthase (4)

Indole-3-glycerol & 1:- !:- C H S P W z

phosphate td

trp3 =TSAI J- Tryptophan Synthase a (5)

Indole a ti

trp2 or orp1,2 J- = TSBl

Tryptophan Synthase p (6)

CH2-CH-COOH

Tryptophan a ?Hz

H

Figure 2. The Tryptophan Biosynthetic Pathway.

Pathway intermediates and genes with mutations are shown on the left, and enzyme names and step numbers (in parentheses) on the right. CdRP, l-(Ocarboxyphenylamino)-l-deoxy-ribulos~5-phosphate.

Tryptophan Biosynthesis 923

Table 1. Plant Tryptophan Biosynthetic Enzyme Loci

Structural Mutant Enzvme Genea Designationb Map Position Reference

Anthranilate synthase a Subunit ASA 1

ASA2

D Subunit

AMT-1

ASBl

AS82 AS83

PR-anthranilate PA T1 transferase

PR-anthranilate PAI1 isomerase PAI2

PAI3

Indole-3-glycerolp hosphate

Tryptophan synthase a synthase lGSl

and p a-Subunit TSA 1

P-Subunit TSB 1

TSB2

TSBl (maize)

TSBP (maize)

amt-1

trp4

trp 1

antisense

trp3

trp2

orp 7

orp2

Chromosome 5, near RFLP 3837

Chromosome 2, between er and RFLP 21502

Linked to ASAl

Chromosome 1, 14.9 cM from GAPB and 21.6 cM from chl l

Chromosome 5, near Ify

Chromosome 5, between RFLPs pCIT1243 and pClTll6

Chromosome 1, near m488 Chromosome 5, near ASAl Chromosome 1, between 93786

and GAPB

Chromosome 3, near BGLl

Chromosome 5, near RFLP 4130

Chromosome 4, near RFLP 1At272

Chromosome 4, near centromere

Chromosome 10, near centromere

Niyogi and Fink (1992)

Niyogi and Fink (1992)

Kreps and Town (1992); C.D. Town (personal communication)

Niyogi et al. (1993)

Niyogi et al. (1993) Niyogi et al. (1993) Last and Fink (1988); Rose et al.

(1 992) Li et al. (1995b) Li et al. (1995b) J. Bender and G. Fink

(unpublished data)

Li et al. (1995a)

E.R. Radwanski, A. J. Barczak, and R.L. Last (unpublished data)

Last et al. (1991); Barczak et al.

Last et al. (1991)

Wright et al. (1992)

(1 995)

Wright et al. (1992)

a All genes are from Arabidopsis unless otherwise noted.

suppression. Those genes for which mutant alleles have been identified are indicated. Antisense indicates that a mutant has been created by antisense

of cDNA clones has facilitated the production of polyclonal antibodies against five of the seven proteins (Zhao and Last, 1995). These antibodies are proving useful for probing the expression of genes involved in the pathway as well as for biochemical analysis of mutants defective in the various pathway steps (Barczak et al., 1995; Radwanski et al., 1995).

Anthranilate Synthase

As is common for committing enzymes in many biosynthetic pathways, anthranilate synthase (EC 4.1.3.27), which converts chorismate and an amino donor (usually glutamine) to anthranilate, appears to be a key regulated enzyme. Because the tryptophan pathway produces a variety of indolic secondary

metabolites, the genetic and biochemical regulation of this enzyme is under active study. At least one form of anthranilate synthase was demonstrated to be feedback inhibited by micromolar concentrations of tryptophan in all plants examined (reviewed in Poulsen and Verpoorte, 1991). The enzymes from cell cultures of the alkaloid-producing plants C. roseus (Poulsen et al., 1993) and Ruta graveolens (Bohlmann et al., 1995) were each purified 4500-fold. Although crude extracts had been reported to contain multiple isoenzymes, only a single form of the purified enzyme was detected from R. graveolens (Bohlmann et al., 1995). Furthermore, although two tryptophan- sensitive forms were resolved during intermediate steps in the purification of the C. m e u s enzyme, these were interpreted as representing conformers rather than isoenzymes (Poulsen and Verpoorte, 1991). The C. m e u s anthranilate synthase has

924 The Plant Cell

Figure 3. Tryptophan Pathway Mutants Are Resistant to 6-Methyl-anthranilate.

See Li et al. (1995b) for details about growth conditions and inhibitorconcentrations.(A) Selection for 6-methylanthramlate-resistant mutants in M2 seed.Two 6-methylanthranilate-resistant plants, with green expanded trueleaves, are surrounded by smaller dying 6-methylanthranilate-sensi-tive plants, with chlorotic cotyledons.(B) Rescreen of true-breeding trpl prototrophic mutant progeny. 6-Meth-ylanthranilate resistance appears uniformly among true-breedingself-cross progeny of the original isolates. The resistant plants are leakytrpl prototrophic mutants defective in PR-anthranilate transferaseactivity.

a K, of 2.6 to 4.7 uM for tryptophan, similar to that reportedfor crude or partially purified anthranilate synthase prepara-tions from other plants (Poulsen et al., 1993).

Biochemical and molecular studies of microbial and plantanthranilate synthase indicate that it is a heterosubunit enzyme

(Crawford, 1989; Niyogi and Fink, 1992; Niyogi et al., 1993;Bohlmann et al., 1995). In microbes, it is well documented thatthe larger a component is responsible for the key enzymaticand allosteric functions. More specifically, anthranilate syn-thase a binds chorismate and catalyzes its aromatization, canuse ammonium as an amino donor in the absence of the 3subunit, and possesses the allosteric tryptophan binding site(Crawford, 1989). In contrast, anthranilate synthase (3 acts asan accessory protein that provides glutamine amidotransfer-ase activity.

cDNAs for two nonallelic anthranilate synthase a subunitshave been identified from both Arabidopsis (ASA1 and ASA2;Niyogi and Fink, 1992) and R. graveolens (ASal and ASo.2;Bohlmann et al., 1995) using a phylogenetically conserved frag-ment of the yeast gene. The predicted Arabidopsis proteinsare 67% identical to one another, whereas R. graveolens ASa1is 78% identical to Arabidopsis ASA1 (and 73% identical toASA2) and R. graveolens ASa2 is 82% identical to Arabidop-sis ASA2 (and 78% identical to ASA1) (Niyogi and Fink, 1992;Bohlmann et al., 1995). Expression of anthranilate synthasea cDNAs from both plants (ASA1, ASal, and ASa2) allowedgrowth of an anthranilate synthase a-deficient E. coli mutanton high concentrations of NH4CI. This demonstrates that, asin prokaryotes, the plant a subunit by itself can use ammo-nium as a substrate (Niyogi and Fink, 1992; Niyogi et al., 1993;Bohlmann et al., 1995).

There is evidence that the a subunit genes are differentiallyregulated during plant development and in response to en-vironmental stimuli. For example, the steady state level ofArabidopsis ASA1 mRNA in rosette leaves is 10-fold higher thanthat of ASA2 and is regulated in a tissue-specific manner,whereas ASA2 mRNA is present at low levels in most tissues(Niyogi and Fink, 1992). Furthermore, accumulation of ASA1mRNA is induced by pathogenesis and wounding, whereasASA2 does not respond to these environmental stresses. Simi-larly, in R. graveolens cultured cells, ASa1 mRNA is induced100-fold after 6 hr of treatment with a fungal elicitor, whereasASa2 is expressed constitutively (Bohlmann et al., 1995). Sur-prisingly, the anthranilate synthase enzyme activity rises onlyfourfold over 12 hr, suggesting that post-transcriptional con-trol mechanisms are important in regulating anthranilatesynthase expression in these elicitor-treated cultured cells.

Experiments performed with E. coli suggest that the plantanthranilate synthase a subunit can interact effectively withthe E. coli anthranilate synthase P subunit (Niyogi and Fink,1992; Bohlmann et al., 1995). Arabidopsis ASA1 and R. grav-eolens ASo.1 and ASa2 all allowed growth of the anthranilatesynthase a-deficient Afrp/E5 mutant on low NH4CI, a condi-tion under which the a subunit is expected to require theglutamine amidotransferase activity of the p subunit (Niyogiand Fink, 1992; Bohlmann et al., 1995). As predicted, growthof an E. coli mutant deleted for both anthranilate synthasesubunit genes (AfrpDE27) was not supported by expressionof cDNAs for either ASA1 or ASa1. However, a surprising re-sult was obtained when the R. graveolens ASa2 cDNA wasexpressed in the E. coli double mutant: despite the absense


of an anthranilate synthase p subunit, the Asa2 protein permitted growth on low ammonium (Bohlmann et al., 1995). This result suggests that the R. gfaveolensASa2 protein either possesses an intrinsic glutamine amidotransferase activity or can interact productively with other E. coli amidotransferases (see the following discussion of subunit associations).

Recent biochemical and molecular cloning results have demonstrated that, as has been shown for the microbial subunits, the plant anthranilate synthase p subunit forms a productive complex with the plant anthranilate synthase a subunit. Bohlmann et al. (1995) found that the R. graveolens a-P complex was stable during purification of anthranilate synthase activity and that the ratio of a to p activity remained constant. In a complementary molecular genetic approach, Niyogi et al. (1993) cloned Arabidopsis anthranilate synthase p cDNAs by functional complementation in €. coli. In this case, p subunit cDNAs were obtained by selecting for clones that produce an amidotransferase activity, which can act in con- cert with Arabidopsis ASA1 to permit growth of the E. coli AtrpDE27double mutant on low ammonium. Genomic gel blot hybridization analysis with an AS67 cDNA probe indicated the presence of three nonallelic genes, which were cloned and partially sequenced. Sequence analysis of the AS67 cDNA revealed a protein coding region with a calculated molecular mass of 30.5 kD, including a presumed plastid target sequence. In contrast to the distinctly different sizes of the a (large) and p (small) subunits from Arabidopsis and microbes, the migra- tion of the R. graveolens subunits is very similar on SDS- polyacrylamide gels (estimated molecular masses of 61 and 62 kD; Bohlmann et al., 1995; see later discussion of subunit associations).

Although a number of 5-methyltryptophan-resistant plant mutants have been previously identified (Widholm, 1976; Ranch et al., 1983; Wakasa and Widholm, 1987; Lee and Kameya, 1991; Kang and Kameya, 1993), some of which could poten- tially result from lesions in anthranilate synthase, to our knowledge the Arabidopsis a-methyltryptophan resistance al- lele amt-7 is the only plant mutation conferring tryptophan analog resistance that has been demonstrated to cause a heritable alteration in anthranilate synthase activity (Kreps and Town, 1992). Crude extracts from amt-7 plants contain approx- imately threefold the amount of anthranilate synthase activity of extracts from wild-type plants, and this activity is approxi- mately twofold less sensitive to feedback inhibition by tryptophan than is wild-type anthranilate synthase (Kreps and Town, 1992). Consistent with the obsewed biochemical phenotype, amt-7 mutant plants contain elevated levels of soluble tryptophan (Kreps and Town, 1992). Such a mutant with re- laxed biochemical regulation of flux through the tryptophan pathway may be expected to have changes in indolic secondary metabolism. Indeed, a 5-methyltryptophan-resistant Lemna gibba variant displays enhanced accumulation and catabolism of free indole-3-acetic acid (IAA) (Tam et al., 1995; see later discussion).

Arabidopsis ASB-deficient mutants (trp4) have been isolated as second-site suppressors that reduce blue fluorescence of a mutant defective in the PR-anthranilate transferase (Niyogi

et al., 1993). This strategy was based upon the expectation that mutations reducing the synthesis of anthranilate would lower the accumulation of blue fluorescent anthranilate and anthranilate P-glycoside in tp7-100 mutants. The screen proved very sensitive to modest reductions in anthranilate synthase activity. In fact, the trp4 mutations were shown to reside in ASB7, which is one of three expressed ASB genes (Niyogi et al., 1993). DNA sequence analysis indicated that the trp4-7 lesion causes the conversion of an absolutely conserved glycine to a gluta- mate and that the trp4-2 mutation affects the 3' splice site of the penultimate intron. Interestingly, reciproca1 genetic crosses revealed a segregation distortion that reduces the transmis- sion of the trp4-7ltrp4-7;trp7-700/trpl-700 double mutation through the female parent. This suggests a possible role for tryptophan, or for secondary metabolites derived from this pathway, in female gametophyte function.

Conversion of Anthranilate to Indole-3-Glycerolphosphate

In contrast with anthranilate synthase, the plant enzymes catalyzing the subsequent three steps in tryptophan biosynthesis (PR-anthranilate transferase, PR-anthranilate isomerase, and indole-3-glycerolphosphate synthase; Figure 2) are just now beginning to be characterized in detail. PR-anthranilate transferase catalyzes the transfer of a phosphoribosyl moiety from phosphoribosylpyrophosphate to anthranilate (Kirschner et al., 1987). In the next (and essentially irreversible) step, PR- anthranilate isomerase rearranges PR-anthranilate to 140- carboxyphenylamino)-l-deoxyribulose-5-phosphate (CdRP) (Kirschner et al., 1987). Indole-3-glycerolphosphate synthase next forms the indole ring during the nearly irreversible conversion of 1 -(Ocarboxy pheny Iam i no)-i -deoxyri bu losed-p hosp hate to indole-3-glycerolphosphate. Because the tryptophan biosynthetic pathway appears to be the only biological source of this important ring structure, this trio of enzymes plays a central role in indolic secondary metabolism.

To our knowledge, none of these three enzymes has yet been purified to homogeneity. However, early studies reported the detection of anthranilate synthase, PR-anthranilate transferase, PR-anthranilate isomerase, indole-3-glycerolphosphate synthase, and tryptophan synthase activities in crude extracts of cultured carrot cells (Widholm, 1973) and of anthranilate synthase, PR-anthranilate transferase, PR-anthranilate isomerase, and indole-3-glycerolphosphate synthase activities in fraction- ated extracts from pea and maize (Hankins et al., 1976). The ability to separate the individual enzyme activities provided an early suggestion that, unlike many microbial enzymes, the plant enzymes are present as separate monofunctional proteins. This hypothesis has been confirmed by more recent molecular analysis.

PR-Anthranilate Transferase

Compared with the biochemical characterization of plant PR- anthranilate transferase (EC 2.4.2.18), the molecular genetic

926 The Plant Cell

analysis of the Arabidopsis enzyme is relatively advanced. The isolation of PAT7 cDNAs was accomplished by complement- ing an E. coli trpD- mutation with an Arabidopsis cDNA expression library (Elledge et al., 1991). In contrast to the other tryptophan biosynthetic structural genes, which are encoded by multiple genes, PR-anthranilate transferase appears to be encoded by a single gene (Rose et al., 1992). Mutation analysis revealed a wealth of phenotypic characteristics associated with PR-anthranilate transferase deficiency in Arabidopsis. For example, all trpl mutants identified to date are resistant to methylanthranilate compounds, and all but one is blue fluorescent under UV light due to the accumulation of anthranilate compounds (Last and Fink, 1988; Rose et al., 1992; A.B. Rose and R.L. Last, unpublished results). A variety of trp7 alleles has been identified by screening for blue fluorescence or methylanthranilate resistance, and the collection of mutants includes both leaky tryptophan prototrophs and severely af- fected auxotrophs. The auxotrophs are especially interesting because they are small and manifest an array of morphologi- cal abnormalities, such as reduced apical dominance and slow growth, that is consistent with a defect in auxin biosynthesis, metabolism, or signal reception (Last and Fink, 1988). Unfor- tunately, these mutants are nearly infertile, making them difficult to propagate. Severa1 weak and strong trp7 alleles have been sequenced; three are missense mutations, and one has both a missense mutation and a single nucleotide change in an intron (A.B. Rose and R.L. Last, unpublished results). Be- cause of the ability to select and screen against wild-type function (methylanthranilate resistance and blue fluorescence, respectively), PAT7 may prove a useful genetic marker in plants.

PR-Anthranilate lsomerase

Except for early reports of PR-anthranilate isomerase activity in crude plant extracts (Widholm, 1973; Hankins et al., 1976), plant PR-anthranilate isomerase has not been characterized biochemically. Molecular biological characterization began with the cloning of two different Arabidopsis PR-anthranilate isomerase cDNAs by complementation of an E. mIi trpC9830 mutation (Li et al., 1995b). High-stringency genomic DNA gel blot hybridizations detected three homologous PAI genes in Arabidopsis. Sequencing of genomic clones for all three genes led to the startling conclusion that the PAI7 and PA12genes are virtually identical, including introns and nontranscribed flanking sequences, and both are very similar to PAI3. All three genes are transcribed in rosette leaves. The discovery of three functional PAIgenes explains why no PR-anthranilate isomerase- deficient mutants were isolated from severa1 hundred thousand M2 Columbia (COLO) and Landsberg erecta (Ler) seedlings screened for blue fluorescence under UV light or selected for anthranilate analog resistance (J. Li and R.L. Last, unpublished results). To examine the effect of a deficiency in PR-anthranilate isomerase, transgenic Arabidopsis plants were created that express PAI7 cDNA in the antisense orientation. The line that was analyzed in detail possessed 10 to 15% of wild-type

PR-anthranilate isomerase protein and enzyme activity and was demonstrated to be both blue fluorescent and 6-methylanthranilate resistant, phenotypes expected for a mutant defective in PR-anthranilate isomerase (Li et al., 199513).

In contrast with the lack of PR-anthranilate isomerase- deficient genetic mutants in the COLO and Ler ecotypes, a blue fluorescent mutant of the Wassilewskija ecotype has recently been identified that has both genetic and epigenetic defects in multiple genes encoding PR-anthranilate isomerase (J. Bender and G. Fink, personal communication). The Wassilewskija ecotype contains four PAI genes rather than the complement of three expressed genes in Col-O; in the pai mutant, two tan- demly repeated PAI genes are deleted. The fluorescence phenotype of the pai mutant, unlike that of the trp7 mutants, is unstable, resulting in both nonfluorescent somatic Sectors and revertant progeny. This phenotypic instability correlates with changes in cytosine methylation and expression of one of the remaining PAIgenes. In nonfluorescent revertant plants, this PAI gene is hypomethylated, and its expression is increased relative to that in fluorescent plants. Because silencing of this PAI gene can be lost in plants with reduced PAI gene copy number, PAI gene silencing may represent a mechanism for mitigating the effects of the inappropriately high gene dosage that results from PAI gene duplication.

The virtual identity of the COLO PAI7 and PAI2 genes raises questions about their origin and function, especially in light of the low level of conservation exhibited by prokaryotic PR- anthranilate isomerase enzymes. Intriguingly, the wide variation seen in prokaryotic PR-anthranilate isomerase is in striking contrast to the much higher level of conservation of bacterial indole-3-glycerolphosphate synthases, a structurally similar eightfold a-b barrel that catalyzes the next reaction (Crawford, 1989). Given that PR-anthranilate isomerase catalyzes a simple rearrangement that can occur nonenzymatically under mild conditions, Crawford (1989) speculated that selective pres- sure against partially inactivating lesions in the PAI gene would have been relatively mild, permitting the acquisition of com- pensatory second-site mutations and resulting in extensive polymorphism. Thus, the striking conservation of the Arabidop sis PAI genes points to a very recent duplication event or to a mechanism that actively maintains the conservation. Genes that have recently undergone duplication are sometimes found in a tandem array on the chromosome (see references cited in Li et al., 1995b); however, the triplicate PAI genes are not clustered in the Arabidopsis genome (Table 1).

Indole-3-Glycerolphosphate Synthase

Complementation of an E. coli mutation (trpC9800) with an Arabidopsis cDNA expression library has allowed the isolation of cDNAs encoding indole-3-glycerolphosphate synthase (EC 4.1.1.48; Li et al., 1995a). The predicted sequence of the Arabidopsis protein shares 22 to 38% identity with microbial proteins. With the increasing attention paid to the possible importance of indole3-glycerolphosphate as a precursor to the


plant hormone IAA in Arabidopsis and maize (Wright et al., 1991; Normanly et al., 1993), more detailed study of this enzyme is warranted.

Tryptophan Synthase a

Tryptophan synthase a is one of two subunits comprising tryptophan synthase (EC 4.2.1.20), the best characterized of the prokaryotic tryptophan biosynthetic enzymes (Bentley, 1990; Miles, 1991). The tryptophan synthase complex catalyzes the last two steps of the pathway: tryptophan synthase a catalyzes the conversion of indole-3-glycerolphosphate to indole, and tryptophan synthase p catalyzes the production of tryptophan from indole and serine. In contrast with the extensive data available for the bacterial and funga1 tryptophan synthase a subunits, the molecular biological investigation of plant tryptophan synthase a is in its infancy. To date, plant TSA sequences are available from maize (V.C. Kramer, L. Artim-Moore, and M.G. Koziel, unpublished data; GenBank accession number "3) and Arabidopsis (TsA7; Radwanski et al., 1995). The Arabidop- sis TsA1 cDNA was isolated by means of a variation on the functional complemetation strategy. An E. coli strain lacking the bacterial genes encoding tryptophan synthase a and tryptophan synthase p but expressing the Arabidopsis 73B7 cDNA was transformed with an Arabidopsis cDNA library. The resulting transformant contained the 73A7 cDNA(Radwanski et al., 1995). The protein inferred from the Arabidopsis TSA1 cDNA contains all of the phylogenetically conserved amino acids known to be essential for microbial tryptophan synthase acata- lytic activity (Miles, 1991). The predicted protein is 34 to 38% identical with the microbial proteins and 53% identical with the predicted maize protein. It is not yet clear whether tryptophan synthase a is encoded by a multigene family, because although genomic DNA gel blot hybridizations at varying strin- gencies detected only one sequence in the Arabidopsis genome that hybridizes strongly to the TsAl cDNA, there are related sequences that are as yet uncharacterized. TSA7 is expressed in wild-type Arabidopsis leaves as a 1.4-kb mRNA and a 30-kD protein, sizes expected for a monofunctional tryptophan synthase a based upon data for microbial enzymes (Radwanski et al., 1995; Zhao and Last, 1995).

Heritable mutations that affect plant tryptophan synthase a activity have only recently been isolated. A stable Hyoscya- mus muficus cell culture variant was proposed to be deficient in tryptophan synthase a based on several indirect lines of evidence (Fankhauser et al., 1990), including rescue by indole but not anthranilate, the accumulation of indolics, the pres- ente of tryptophan synthase p activity, and the lack of overall tryptophan synthase activity (Figure 2, steps 5 and 6). Unfor- tunately, plants were not regenerated from this variant. Selection of ethyl methanesulfonate-mutagenized Arabidop- sis M2 seeds on 5-methylanthranilate led to the isolation of two genetically inherited tryptophan synthase a deficien- cies. These mutants (trp3) accumulate reduced amounts of tryptophan synthase a mRNA and have very little tryptophan

synthase a cross-reactive protein (Zhao and Last, 1995; E.R. Radwanski and R.L. Last, unpublished data).

Tryptophan Synthase p

The tryptophan synthase p subunit is more phylogenetically conserved than the other tryptophan pathway enzymes (Miles, 1991). This allowed the cloning of the Arabidopsis 73B7 gene by screening Arabidopsis libraries with a yeast TffP5 gene probe (Berlyn et al., 1989). Using 73B7 to probe genomic DNA gel blots showed that Arabidopsis possesses a similar gene, EB2, which encodes a protein that is >95% identical to TSBl (Last et al., 1991). cDNA clones for two nonallelic maize genes were then identified based on their homology to the Arabidopsis 73B1 cDNA; the predicted maize proteins, TSB1 and TSB2, share 98% identity and are 89 and 83% identical, reSpectiVely, to TSBl from Arabidopsis (Wright et al., 1992).

Although the existence of functional duplicate genes might be expected to complicate or prevent the isolation of mutants, tryptophan synthase P-deficient mutants have been identified in both Arabidopsis and maize (Last et al., 1991; Wright et al., 1992). The Arabidopsis trp2-7 mutation, which decreases tryptophan synthase p activity to 4 5 % of wild-type levels, was mapped to the more highly expressed TSB1 gene (Last et al., 1991). This result indicates that 7362 is not sufficient to con- fer tryptophan-independent growth under normal growth conditions. In contrast, a tryptophan-dependent phenotype in maize requires the inactivation of two TSB genes (orange pericarpl = ORP 1 = E67 and orange pericarp2 = Off P 2 = TSB2). The orpl;ofp2 double mutant was first studied because it causes the filial expression of an orange coloration in the maternally inherited pericarp tissue (Wright and Neuffer, 1989; Wright et al., 1992). A defect in the tryptophan pathway was suggested by the strong indole odor and the accumulation of blue fluorescent compounds in the double mutant seedlings. The orange pericarp coloration is apparently due to the oxida- tion of indole that accumulates and diffuses from the endosperm into the pericarp. Restriction fragment length polymorphism (RFLP) analysis demonstrated that the maize orp loci coseg- regate with the two nonallelic 738 genes (7361 and TS62), suggesting that the orp loci are duplicate structural genes (Wright et al., 1992).

The observation that trp2-7 plants are resistant to B-fluoroindole (presumably due to reduced conversion of Mluoroindole to toxic 5-fluorotryptophan) indicated that tryptophan synthase- deficient Arabidopsis mutants can be selected directly with this analog (Last et al., 1991). This selection yielded a trp2 allelic series with varying amounts of E67 mRNA and protein accumulation (Barczak et al., 1995). DNA sequence analysis of three new alleles confirmed the existence of mutations in 73B1. Analysis of this allelic series revealed several interesting aspects of pathway regulation. One is a correlation between the leve1 of accumulation of mutant tryptophan synthase p proteins and wild-type tryptophan synthase a protein. This result suggests either that the synthesis of these two subunits (which

928 The Plant Cell

presumably function in a prokaryote-like heterotetramer) is coordinated or that the p subunit influences the stability of the a subunit. Second, the trp2 mutants all accumulate repro- ducibly higher levels of anthranilate synthase a subunit protein, suggesting that the plant responds to tryptophan limitation by increasing the amount of the committing enzyme for the tryptophan biosynthetic pathway.

Subunit Associations in the Plant Enzymes

The existence in microbes of fusion proteins containing two to three catalytic domains in various combinations (Hütter et al., 1986; Crawford, 1989) appears to have no parallel in the characterized plant tryptophan biosynthetic enzymes. The plant enzymes are all monofunctional, based upon cDNA and genomic sequences and on the sizes of transcripts and proteins (Berlyn et al., 1989; Last et al., 1991; Niyogi and Fink, 1992; Rose et al., 1992; Wright et al., 1992; Niyogi et al., 1993; Li et al., 1995a, 1995b; Radwanski et al., 1995; Zhao and Last, 1995). Nearly all of the plant proteins that have been analyzed are the sizes predicted from studies of the microbial domains. One exception is the larger anthranilate synthase p subunit of R. graveolens (the purified protein is >60 kD; Bohlmann et al., 1995) and of Dianrhus caryophyllus (the partially purified D subunit of anthranilate synthase is ~ 7 0 kD; Matern, 1994). Monofunctional prokaryotic p subunits range in size from 15 to 24 kD (Zalkin, 1980), and some plant p subunits are close to that size: anthranilate synthase p purified from C. roseus is 25 kD (Poulsen et al., 1993), and the predicted product of the Arabidopsis AS67 locus is 30.5 kD (Niyogi et al., 1993). However, there are a number of w6O-kD multifunctional microbial enzymes containing anthranilate synthase p activity (Zalkin, 1980). Large p subunits are found in plants that produce secondary metabolites directly from anthranilate. It is interesting to speculate that these may be multifunctional enzymes that channel substrates into the production of tryptophan or secondary metabolites such as anthranilate-derived al kaloids.

The monofunctionality of the plant tryptophan biosynthetic proteins does not mean that they act alone. Indeed, it may give them the flexibility to engage in multiple interactions with other proteins. One notable example of such interactions is the synthesis of paminobenzoate in prokaryotes. Whereas chorismate and glutamine can be transformed into anthranilate (o-aminobenzoate) by the action of anthranilate synthase in the committing step of tryptophan biosynthesis, they can also be converted into paminobenzoate in a similar reaction catalyzed by p-aminobenzoate synthase (Goncharoff and Nichols, 1984; Green and Nichols, 1991). In some prokaryotes, a single p subunit acts as glutamine amidotransferase in both anthranilate synthase and paminobenzoate synthase (Crawford, 1989). In avariation on this theme of shared subunits, the R. graveolens Asa2 protein exhibits glutamine amidotransferase activity in an E. coli double mutant that is deficient in both the a and p subunits of anthranilate synthase, raising the possibility that

the plant ASA2 (ASa2) protein may interact productively with E. coli amidotransferases other than anthranilate synthase p (Bohlmann et al., 1995). The E. colienzymes anthranilate synthase, paminobenzoate synthase, and isochorismate synthase, all of which use chorismate as a substrate, are closely related, with -40% similarity in their C-terminal domains (Ozenberger et al., 1989). lsochorismate synthase activity has been demonstrated in crude extracts of higher plants, where it is involved in phylloquinone (vitamin K,) and anthraquinone synthesis (Poulsen et al., 1991). To our knowledge, paminobenzoate synthase activity has not been reported in plants. It will be interesting to learn whether plant anthranilate synthase, paminobenzoate synthase, and isochorismate synthase share subunits.

Another open question is whether other chorismate-utilizing enzymes are involved in the synthesis of aromatic secondary metabolites in plants. A microbial example is provided by the phenazine pigment-producing bacterium Pseudomonas aer- uginose. This species possesses two highly related sets of anthranilate synthase enzymes, encoded by trpE trpG and phnA phn6. Although the phnA phn6 gene pair is primarily responsible for the synthesis of the blue-green phenazine pigment pyocyanin from chorismate, it is able to complement E, coli trpE and trp€(G) mutations (Essar et al., 1990).

Tryptophan synthase is another example of a multimeric enzyme whose complex subunit interactions have been extensively studied in microbes. The a2fi2 heterotetrameric complex forms a classic channeling enzyme: the intermediate, indole, moves from the a subunit active site to the p subunit active site through a 25-A-long tunnel (Hyde et al., 1989). The p subunit activity in turn converts indole and L-serine into tryptophan. The bacterial tryptophan synthase a and tryptophan synthase p subunits are intimately associated and require each other for full enzyme activity even in their separate reactions (Crawford, 1989; Miles, 1991). In contrast to the synthesis of separate subunits in bacteria, fungi such as Sacchammyces cerevisiae and Neurospora crassa produce active tryptophan synthase as a dimer of a single polypeptide that contains fused a and p domains (Matchett and DeMoss, 1975; Dettwiler and Kirschner, 1979; Zalkin and Yanofsky, 1982; Burns and Ymofsky, 1989). Despite the solid foundation provided by the extensive work on the microbial enzymes, our understanding of the structure and activity of this plant enzyme is still primitive.

Severa1 lines of evidence suggest that the plant tryptophan synthase resembles the prokaryotic complex of a and p subunits. Tryptophan synthase activity in partially purified preparations from pea (Nagao and Moore, 1972) and tobacco (Delmer and Mills, 1968) can be separated into two compo- nents analogous to prokaryotic a and p activities, and both are required for full activity in the overall reaction (Figure 2, steps 5 and 6). An Arabidopsis cDNA expression library failed to rescue a tryptophan synthase a-deficient (frpA) mutant of E. coli, but rescue was successful when an active Arabidop- sis tryptophan synthase p protein was also expressed, demonstrating that the presence of plant tryptophan synthase p protein activates the plant a subunit in E. coli. Consistent


with this genetic result, the Arabidopsis a and p subunits copurify by immunoaffinity chromatography (Radwanski et al., 1995).

Although there is evidence that plant anthranilate synthase and tryptophan synthase are active as a-P complexes, it is not known whether other plant tryptophan biosynthetic proteins interact physically with each other and/or with these synthase complexes, perhaps to channel unstable substrates to the next active site or to stabilize active protein conforma- tions. There are suggestions in the literature that such complexes may be possible in organisms other than higher plants. For example, the PR-anthranilate transferase, PR- anthranilate isomerase, indole-3-glycerolphosphate synthase, and tryptophan synthase activities copurify from the photosyn- thetic unicellular eukaryotic alga Euglena gracilis (Lara and Mills, 1972). Additionally, the trifunctional enzyme of the fungus N. crassa, containing anthranilate synthase, indole-3-glycerolphosphate synthase, and PR-anthranilate isomerase activities, is a dimer arranged to make the product of the PR-anthranilate isomerase-catalyzed reaction a more effective substrate for the indole-3-glycerolphosphate synthase active site than is exogenous substrate (Gaertner et al., 1970).

However, there are also reports suggesting that such complexes need not exist. These include the unsuccessful search by Hommel et al. (1989) for a complex of the yeast monofunctional PR-anthranilate transferase and PR-anthranilate isomerase enzymes that would allow channeling of the unstable and energetically expensive intermediate PR-anthranilate. Another example is provided by x-ray crystallographic data demonstrating that the indole-3-glycerolphosphate synthase and PR-anthranilate isomerase active sites face away from each other in the E. coli bifunctional enzyme. This suggests that CdRP moves through the cytosol to the indole-3- glycerolphosphate synthase active site (Kirschner et al., 1987). Although the plant tryptophan biosynthetic enzymes presumably all reside within the chloroplast stroma (see later discussion), it is not known whether the unstable intermediates diffuse from one active site to another or whether the enzymes are associated in an assemblage reminiscent of the E. gracílis multifunctional complex (Lara and Mills, 1972; Hrazdina and Jensen, 1992).

Regulation of the Tryptophan Biosynthetic Pathway in Plants

Microorganisms are known to regulate tryptophan biosynthesis both by modulating gene expression and by allosteric regulation of anthranilate synthase by tryptophan. Bacteria, which must respond quickly to extreme shifts in environmental conditions, regulate tryptophan production by modulating gene expression at severa1 levels. These mechanisms include tryptophan-mediated repression of trp operon transcription, induction of frpsA (tryptophan synthase) transcription by the substrate indole-3-glycerolphosphate, coupling of translation and transcription (attenuation), and coupled translation of frpED (anthranilate synthase and PR-athranilate transferase) and

trpBA (Yanofsky and Crawford, 1987). In contrast to the specific regulation of tryptophan biosynthesis operating in bacteria, yeast respond to starvation for single amino acids by derepress- ing transcription of biosynthetic enzymes in multiple pathways (Jones and Fink, 1982; Hinnebusch, 1992). This general control network is mediated by the DNA binding protein GCN4, the synthesis of which is translationally regulated (Hinnebusch, 1994). This strategy is reminiscent of bacterial attenuation in that both allow the translational state of the cell to influence transcription of amino acid biosynthetic enzymes.

As sessile organisms, higher plants have evolved nove1 strategies to accommodate the demands for amino acids due to growth, development, and environmental stresses. Unlike the tryptophan biosynthetic pathways of most bacteria and fungi, the plant pathway is more than a housekeeping pathway for production of a component of protein synthesis. It is also the source of precursors for highly inducible defense- related secondary metabolites and for the hormone IAA, which is present in extremely low concentrations. Given the variety of situations in which the plant might need to fine-tune the synthesis of tryptophan or pathway intermediates, it would not be surprising if plants employ regulatory strategies not found in microbes.

Published results indicate the importance of anthranilate synthase in regulation of the plant pathway. This enzyme is allosterically inhibited by micromolar tryptophan (Widholm, 1972; Poulton and Moller, 1993), and elevated levels of free tryptophan are detected in 5-methyltryptophan-resistant mutant plant cell cultures and calli, and in plants possessing an anthranilate synthase with decreased sensitivity to inhibition by tryptophan (reviewed in Poulsen and Verpoorte, 1991). In addition, accumulation of anthranilate synthase mRNA is induced under stress conditions such as wounding, pathogen infection, and funga1 elicitation (Niyogi and Fink, 1992; Niyogi et al., 1993; Bohlmann et al., 1995). Anthranilatesynthase protein leve1 and enzyme activity also appear to be influenced by the tryptophan biosynthetic capacity of Arabidopsis plants. lmmunoblots of protein extracts from leaf tissue of an allelic series of frp2 mutants revealed a two- to fivefold increase in the amount of anthranilate synthase a protein over that detected in wild-type plants (Barczak et al., 1995). Additionally, increased anthranilate synthase activity was reported in extracts of trpl-700 (Niyogi et al., 1993) and trp2-l plants (Last et al., 1991). It is possible that the mutant plants respond to reduced ability to synthesize tryptophan by increasing the activity of the committing enzyme. This is an area that deserves further study.

Localization of the Tryptophan Biosynthetic Pathway in Plants

In contrast to early reports of aromatic amino acid biosynthetic isoenzymes with different biochemical properties and subcellular localizations (reviewed in Singh et al., 1991), there is no evidence for a tryptophan biosynthetic pathway outside of ttie

930 The Plant Cell

plastid. On the contrary, sequence analysis of Arabidopsis cDNAs encoding each enzyme in the tryptophan biosynthetic pathway suggests the presence of plastid target sequences at the N terminus of each protein (Last et ai., 1991; Niyogi and Fink, 1992; Rose et al., 1992; Niyogi et al., 1993; Li et ai., 1995a, 1995b; Radwanski et al., 1995). The same is true of the R. grav- eolensASa7 and ASa2 (Bohlmann et al., 1995) and maize TSA (V.C. Kramer, L. Artim-Moore, and M.G. Koziel, unpublished data; GenBank accession number X76713), TSB1, and TSBP (Wright et al., 1992) sequences. More compelling evidence for chloroplastic localization is provided by direct biochemical assays. In vitro import assays have demonstrated the chloroplast uptake of the Arabidopsis PAI1 and TSAl (Zhao and Last, 1995) and the R. graveolensASa7 (Bohlmann et al., 1995) precursor proteins. Furthermore, over 98% of Arabidopsis tryptophan synthase activity resides in a chloroplast-enriched fraction (Last et al., 1991), and immunoblot analysis has demonstrated that virtually all of the Arabidopsis anthranilate synthase a, PR-anthranilate transferase, PR-anthranilate isomerase, tryptophan synthase a, and tryptophan synthase P proteins are found in a chloroplast fraction (Zhao and Last, 1995). Although these data strongly support localization of the pathway to the chloroplast, proving that the chloroplast-localized enzymes are sufficient to produce tryptophan for a11 the needs of the plant cell will require testing in vivo.

TRYPTOPHAN METABOLISM

In plants, products of the tryptophan pathway are incorporated into polypeptides and metabolized into auxin, glucosinolates, phytoalexins, alkaloids, and other indolic compounds. These metabolites play diverse roles in plant biological processes, including development, plant-pathogen interactions, herbivory, and pollination biology. Some of these compounds are also important in producing pharmaceuticals and textiles. Despite the importance of this group of molecules, the biosynthetic pathways for many are largely unknown. As described later, studies using tryptophan biosynthetic mutants have challenged the idea that tryptophan is an obligatory intermediate in the biosynthesis of some indolic secondary products. Furthermore, mutants in indolic secondary metabolism are being characterized, creating exciting research directions. In this section, we review recent ideas on the production of auxin, indolic phytoalexins, and glucosinolates (Figure 1). lnterested readers are encouraged to consult recent reviews on auxin (Normanly et al., 1995) and indolic glucosinolates and phytoalexins (Chapple et al., 1994) for more details.

Indole-3bcetic Acid

It is reasonable to expect that the synthesis of the plant hormone auxin, which exerts strong biological effects at very low concentrations, would be tightly controlled. Recent reports on

the biosynthesis of auxin suggest that different plant species are capable of synthesizing IAA from various pathways originat- ing from either tryptophan or pathway intermediates (Nonhebel et al., 1993). For example, results from labeling Lema gibba with D- and ~-W-tryptophan were inconsistent with any significant biosynthesis of IAA from L- or D-tryptophan (Baldi et al., 1991), whereas labeling experiments using 2H20 (D20) and 2H5-L-tryptophan demonstrated conversion of L-tryptophan to IAA in bean seedlings (Bialek et al., 1992) and undifferen- tiated carrot cell cultures (Michalczuk et al., 1992). However, the latter study also provided evidence that carrot somatic em- bryogenic cells utilize a non-tryptophan pathway (Michalczuk et ai., 1992). These seemingly contradictory results suggest that, during different stages of development, cells of single species may utilize different biosynthetic routes to auxin.

The availability of plant.mutants altered in tryptophan biosynthesis provides opportunities to analyze critically the pathways for auxin production and the role of the IAA turnover rate in plant growth and development. lsotope dilution gas chromatography-selected ion monitoring-mass spectrom- etry was used to demonstrate that the tryptophan-requiring trp3-7 (tryptophan synthase a-deficient) and trp2-7 (tryptophan synthase P-deficient) Arabidopsis mutants accumulate high levels of amide- and ester-linked IAA, pointing to either indole- 3-glycerolphosphate or indole as an auxin precursor, rather than to tryptophan (Normanly et al., 1993). Results of similar labeling experiments in seedling-lethal tryptophan synthase B-deficient maize orpl;orp2 seedlings also pointed to a non- tryptophan pathway for IAA production (Wright et al., 1992).

In addition to demonstrating synthesis of auxin by a tryptophan-independent pathway, labeling experiments have revealed that the Arabidopsis tryptophan auxotrophic mutant seedlings accumulate indole-3-acetonitrile (IAN). This compound, one of many candidates for an auxin biosynthetic intermediate, had been thought to derive from tryptophan. How- ever, Normanly et al. (1993) found that the trp3-7 (tryptophan synthase a-deficient) and trp2-7 (tryptophan synthase P-deficient) mutants accumulate elevated amounts of IAN. These results suggest that Arabidopsis may produce IAN from a tryptophan pathway intermediate such as indole-3-glycerolphosphate. The fate of IAN in vivo is not entirely clear. Although the enzyme nitrilase is able to convert IAN to IAA in vitro, it has variously been dismissed as lacking general importance dueto its limited distribution in plants (Nonhebel et ai., 1993) and proposed to be a key enzyme in IAA production in the Brassicaceae, including Arabidopsis (Bartling et al., 1992). Four Arabidopsis genes for nitrilase have been identified by Bartling et ai. (1992) and Bartel and Fink (1994). The different expression patterns of nitrilase I, a soluble enzyme, and nitrilase 11, a peripheral membrane protein, led to the working hypothesis that nitrilase I functions throughout vegetative development, whereas nitrilase II, which is most abundant in siliques, may provide a biosynthetic route from indole glucosinolates to auxin during seed germination (Bartel and Fink, 1994; Bartling et al., 1994). Analysis of both nitrilase-overproducing transgenic plants and transgenic plants expressing an antisense nitrilase


gene should provide a critical test of the importance of this enzyme in the production of IAA and elucidate the specific roles of the members of this gene family.

lndole Glucosinolates

Glucosinolates are anionic thioglucosides synthesized mainly by members of the order Capparales, including all the Bras- sicaceae (Figure 1; Larsen, 1981). Upon mechanical injury to plant tissue, glucosinolates are hydrolyzed by the enzyme myrosinase to yield mustard oils, which can include isothiocya- nate, organic nitrile, or thiocyanate (Underhill, 1980; Poulton and Moller, 1993). Although the function of these degradation compounds in plants is not clear, they may play a role in defense against attack by bacteria, fungi, and insects. For example, isothiocyanates are toxic to some insects and also exhibit antibacterial and antifungal effects (Underhill, 1980; Fenwick, 1983). Glucosinolates are also known to affect plant- insect interactions, attracting some species of insects to feed or deposit eggs and inhibiting feeding by others (Poulton and Moller, 1993).

The indole glucosinolates are believed to be derived from tryptophan, whereas other classes of glucosinolates are thought to arise from other amino acids (Haughn et al., 1991). It has been proposed that indole glucosinolates may give rise to auxin (Larsen, 1981; Bartling et al., 1994). One of the six Arabidopsis mutants with an altered glucosinolate profile isolated in a brute force screen had both lower indole and aliphatic leaf glucosinolates, whereas none of the mutants showed a change in just the indole glucosinolates (Haughn et al., 1991). Intriguingly, the mutant with decreased indole glucosinolates (TU8) is a dwarf with underdeveloped leaves, and the dwarf phenotype (which is suggestive of a defect in auxin production) cosegregates with the glucosinolate phenotype. Plants defective in indole glucosinolate production specifically are needed to clarify the biological importance of these compounds and the biosynthetic pathway.

Research on the glucosinolates is stimulated by the desire to control the levels of these troublesome compounds in crop plants. Livestock feeding on fodder with a high glucosinolate content like canola meal suffer from dietary problems, and it may be desirable to reduce the strong flavor and odor of the Brassicaceae used as vegetables and condiments (Haughn et al., 1991; Poulton and Moller, 1993). In an attempt to divert tryptophan into tryptamine at the expense of indole glucosinolate production, canola was transformed with the C. roseus tryptophan decarboxylase gene driven by the cauliflower mo- saic virus 35s promoter (Chavadej et al., 1994). In the seven independent transgenic lines analyzed, indole glucosinolate levels were reduced in all tissues examined, and in two lines the seeds had only 3% of control plant values. The diversion of tryptophan away from the indole glucosinolate biosynthetic pathway and into tryptamine production is consistent with the hypothesis that tryptophan is a precursor to the indole glucosinolates.

Phytoalexins

The indole ring is also used in the biosynthesis of certain phytoalexins, low molecular weight antimicrobial compounds, ranging from simple aliphatics to complex terpenoids, that are synthesized by plants in response to infection (Gross, 1993; Dixon and Paiva, 1995, this issue). The Brassicaceae produce indolic phytoalexins substituted at position 2 and/or 3 with nitrogen- and sulfur-containing residues. The indolic phytoalexin camalexin (3-thiazol-2’-yl-indole) was demonstrated to be produced by Arabidopsis inoculated with the bacterial pathogen Pseudomonas syringae pv syringae, which elicits a hypersensitive response (Tsuji et al., 1992). This compound was shown to inhibit pathogen growth in vitro and to accumulate in plants in a pattern negatively correlated with in vivo pathogen growth. Despite the recent availability of camalexin- deficient mutants, the role of this phytoalexin in vivo in the plant response to pathogen attack remains unclear. The mutants are unaffected in the ability to restrict growth of avirulent bacteria, strongly suggesting that camalexin is not required for resistance of Arabidopsis to avirulent F! syringae. One mutant lacking any detectable camalexin exhibited a wild-type response to virulent F! syringae, whereas others, with 10 or 30% of normal camalexin levels, allowed more growth of the virulent strain (Glazebrook and Ausubel, 1994).

Reminiscent of work on auxin biosynthesis, the biosynthetic pathway to camalexin was probed using Arabidopsis tryptophan biosynthetic mutants (Tsuji et al., 1993). Detached wild-type leaves treated with silver nitrate (an abiotic elicitor of camalexin synthesis) were found to incorporate 1 l-fold more 14C-anthranilate into camalexin than untreated controls. However, elicitor-treated and control plants did not differ in the incorporation of 3H-tryptophan into camalexin, contrary to the result expected if tryptophan is a biosynthetic precursor. The observation that the specific activity of 14C-anthranilate-labeled camalexin was five to six times greater than that achieved with 3H-tryptophan is consistent with a tryptophan-independent biosynthetic pathway. In addition, after elicitation, a leaky prototrophic PR-anthranilate transferase mutant blocked early in tryptophan biosynthesis (trpl-100; Figure 2, step 1) contained only 50% of the wild-type leve1 of camalexin, whereas the auxotrophic tryptophan synthase trp3-7 and ffp2-7 mutants (Figure 2, steps 5 and 6) had wild-type levels. These experiments suggest that camalexin is probably made from indole-3-glycerolphosphate rather than from tryptophan (Tsuji et al., 1993) and do not support the earlier speculation that the indole phytoalexins arise from the indole glucosinolates (Takasugi et al., 1988). The analysis of additional biosynthetic mutants coupled with careful isotope dilution studies should help to clarify the exact biosynthetic pathway leading to camalexin.

CONCLUSIONS AND PERSPECTIVES

The merger of genetics, molecular biology, and biochemistry has allowed significant progress in studies of the tryptophan

932 The Plant Cell

biosynthetic pathway of plants. Although the pathway is most intensively studied in Arabidopsis, important information is becoming available in other species, including severa1 alkaloid- producing plants. It is of particular interest to understand how this pathway is regulated, both during normal development and in response to conditions that cause increased demands for synthesis of indolic and anthranilate-derived secondary metabolites. Such information will not only contribute to our understanding of basic biochemical and genetic regulatory mechanisms in plants but should also suggest strategies for increasing the yields of medically important indolic products.

The transport of amino acids, including tryptophan, is a pro- cess that is relatively poorly understood in plants. Although there is good evidence for the importance of transport of the amino acids central to nitrogen metabolism (such as aspara- gine and glutamine; see Lam et al., 1995, this issue), the physiological significance of long-distance movement of tryptophan is unclear. In addition, although tryptophan must move from the plastid (where it is synthesized) to the cytosol and mitochondria for protein synthesis, the mechanisms of interor- ganellar amino acid transport are largely unknown. Plant mutants deficient in tryptophan biosynthesis and transport should be useful tools in studying these problems in amino acid trafficking.

ACKNOWLEDGMENTS

We thank Judith Bender, Jorg Bohlmann, Clint Chapple, Jane Glazebrook, and Jennifer Normanly for critical comments on sections of this review and Katherine Denby for comments on the entire manu- script. Research was funded by grants from the National lnstitutes of Health (NIH) (Grant No. GM43134), Biotechnology Research and De- velopment Corporation (Peoria, IL), and a National Science Foundation (NSF) Presidential Young lnvestigator Award (Grant No. DMB-9058134) to R.L.L. E.R.R. wasfunded in part by fellowships from an NIH Training Grant to the Cornell University Section of Genetics and Development, from the Cornell NSF Plant Science Center, a unit in the U.S. Depart- ment of Agriculture-U.S. Department of Energy-NSF Plant Science Centers Program, and from the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Founda- tion, a consortium of industries, and the U.S. Army Research Office.

REFERENCES

Baldi, B.G., Maher, B.R., Slovin, J.P., and Cohen, J.D. (1991). Sta- ble isotope labeling, in vivo, of D- and L-tryptophan pools in Lemna gibba and the low incorporation of label in indole-3-acetic acid. Plant Physiol. 95, 1203-1208.

Barczak, A.J., Zhao, J., Prultt, K.D., and Last, R.L. (1995). 5-Fluo- roindole resistance identifies tryptophan synthase j3 subunit mutants in Arabidopsis thaliana. Genetics 140, 303-313.

Bartel, B., and Flnk, G.R. (1994). Differential regulation of an auxin-

producing nitrilase gene family in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 91, 6649-6653.

Bartllng, D., Seedorf, M., Mlthoefer, A., and Weller, E.W. (1992). Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone indole-3-acetic acid. Eur. J. Biochem 205, 417-424.

Bartllng, D., Seedorf, M., Schmldt, R.C., and Weller, E.W. (1994). Molecular characterization of two cloned nitrilases from Arabidop- sis thaliana: Key enzymes in biosynthesis of the plant hormone indole-3-acetic acid. Proc. Natl. Acad. Sci. USA 91, 6021-6025.

Rentley, R. (1990). The shikimate pathway-A metabolic tree with many branches. CRC Crit. Rev. Biochem. MOI. Biol. 25, 307-384.

Berlyn, M.B., Last, R.L., and Flnk, G.R. (1989). A gene encoding the tryptophan synthase j3 subunit of Arebidopsis thaliane. Pm. Natl. Acad. Sci. USA 86, 4604-4608.

Blalek, K., Michalczuk, L., and Cohen, J.D. (1992). Auxin biosynthesis during seed germination in Phaseolus vulgaris. Plant Physiol.

Bohlmann, J., De Luca, V., Ellert, U., and Martln, W. (1995). Purifi- cation and cDNA cloning of anthranilate synthase from Ruta g m l e n s : Modes of expression and properties of native and recom- binant enzymes. Plant J. 7, 491-501.

Burns, D.M., and Yanofsky, C. (1989). Nucleotide sequence of the Neurospora crassa tp-3 gene encoding tryptophan synthetase and comparison of the trp3 polypeptide with its homologs in Sac- charomyces cerevisiae and Escherichia coli. J. Biol. Chem. 264,

Chapple, C.C.S., Shlrley, B.W., Zook, M., Hammerschmldt, R., and Somerville, S.C. (1994). Secondary metabolism in Arabidopsis. In Arabidopsis, E.M. Myerowitz and C.R. Somerville, eds (Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press), pp. 989-1030.

Chavadej, S., Brlsson, N., McNell, J.N., and De Luca, V. (1994). Redirection of tryptophan leads to production of low indole glucosine late canola. Proc. Natl. Acad. Sci. USA 91, 2166-2170.

Crawford, I.P. (1989). Evolution of a biosynthetic pathway: The tryptophan paradigm. Annu. Rev. Microbiol. 43, 567-600.

Delmer, D.P., and Mills, S.E. (1968). Tryptophan synthase from Nico- tiana tabacum. Biochim. Biophys. Acta 167, 431-443.

Dettwiler, M., and Kirschner, K. (1979). Tryptophan synthase from Saccharomyces cerevisiae is a dimer of two polypeptide chains of M, 76 O00 each. Eur. J. Biochem. 102, 159-165.

Dlxon, R.A., and Palva, N.L. (1995). Stress-induced phenylpropanoid metabolism. Plant Cell 7, 1085-1097.

Elledge, S.J., Mulllgan, J.T., Ramer, S.W., Spottswood, M., and Davis, R.W. (1991). LYES: A multifunctional cDNA expression vec- tor for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl. Acad. Sci. USA88,1731-1735.

Essar, D.W., Eberly, L., Hadero, A., and Crawford, I.P. (1990). Iden- tification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: lnterchangeability of the two anthranilate synthases and evolutionary implications. J. Bacteriol.

Fankhauser, H., Pythoud, F., and Klng, P.J. (1990). A tryptophan auxe troph of Hyoscyamus muticus lacking tryptophan-synthase activity. Planta 180, 297-302.

Fenwlck, G.R., Heaney, R.K., and Mullin, W.J. (1983). Glucosino- lates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutrit. 18, 123-148.

100, 509-517.

3840-3848.

172, 884-900.


Gaertner, F.H., Ericson, M.C., and DeMoss, J.A. (1970). Catalytic facilitation in vitro by two multienzyme complexes from Neumspora crassa. J. Biol. Chem. 245, 595-600.

Gilchrist, D.G., and Kosuge, T. (1980). Aromatic amino acid biosynthesis and its regulation. In The Biochemistry of Plants, Vol. 5, B.J. Miflin, ed (New York Academic Press), pp. 507-531.

Glazebrook, J., and Ausubel, F.M. (1994). lsolation of phytoalexin- deficient mutants of Arebidopsis thaliana and characterization of their interactions with bacterial pathogens. Proc. Natl. Acad. Sci. USA

Gonchamff, I?, and Nichols, B.P. (1984). Nucleotide sequence of Esch- erichia coli pabB indicates a common evolutionary origin of p-aminobenzoate synthetase and anthranilate synthetase. J. Bac- teriol. 159, 57-62.

Green, J.M., and Nichols, B.P. (1991). p-Aminobenzoate biosynthesis in Escherichie coli. J. Biol. Chem. 266, 12971-12975.

Gmss, D. (1993). Phytoalexins of the Brassicaceae. 2. Pflanzenkr. Pflan- zenschurz 100,433-442.

Hanklns, C.N., Largen, M.T., and Mills, S.E. (1976). Some physical characteristics of the enzymes of L-tryptophan biosynthesis in higher plants. Plant Physiol. 57, 101-104.

Haughn, G.W., Davln, L., Glblin, M., and Underhill, E.W. (1991). Bio- chemical genetics of plant secondary metabolites in Arabidopsis thaliena: The glucosinolates. Plant Physiol. 97, 217-226.

Herrmann, K.M. (1995). The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. Plant Cell 7, 907-919.

lerrmann, K.M., Zhao, J.-M., Pinto, J.E.B.P., Weaver, L., and Henstrand, J.M. (1992). Regulation of carbon flow into the shikimate pathway. In Biosynthesis and Molecular Regulation of Amino Acids in Plants, H. Flores, J. Shannon, and 6. Singh, eds (Rock- ville, MD: American Society of Plant Physiologists), pp. 12-18.

Iinnebusch, A.G. (1992). General and pathway-specific regulatory mechanisms controlling the synthesis of amino acid biosynthetic enzymes in Saccharomyces cerevisiae. In The Molecular and Cel- lular Biology of the Yeast Saccharomyces, Vol. 2, E.W. Jones, J.R. Pringle, and J.B. Broach, eds (Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press), pp. 321-414.

Hinnebusch, A.G. (1994). Translational control of GCN4: An in vivo barometer of initiation-factor activity. Trends BiÓchem. Sci. 19,

Hommel, U., Lustig, A., and Klrschner, K. (1989). Purification and characterization of yeast anthranilate phosphoribosyltransferase. Eur. J. Biochem. 180, 33-40.

Hrazdina, G., and Jensen, R.A. (1992). Spatial organization of enzymes in plant metabolic pathways. Annu. Rev. Plant Physiol. Plant MOI. Biol. 43, 241-267.

Hütter, R., Niederberger, P., and DeMoss, J.A. (1986). Tryptophan biosynthetic genes in eukaryotic microorganisms. Annu. Rev. Microbiol. 40, 55-77.

Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W., and Davies, D.R. (1989). Three-dimensional structure of the tryptophan synthase a& multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 264, 17857-17871.

Jones, E.W., and Fink, G.R. (1982). Regulation of amino acid and nucleotide biosynthesis in yeast. In The Molecular and Cellular Bi- ology of the Yeast Sacchammyces, Vol. 1, J.N. Strathern, E.W. Jones,

91, 8955-8959.

409-414.

and J.R. Broach, eds (Cold Spring Harbor, NY: Cold Spring Harbor Press), pp. 181-299.

Kang, K.K., and Kameya, T. (1993). Selection and characterization of a 5-methyltryptophan resistant mutant in Zea mays L. Euphytica

Kirschner, K., Szadkowski, H., Jardetzky, T.S., and Hager, V. (1987). Phosphoribosylanthranilate isomerase-indoleglycerol-phosphate synthase from €scherichie coli. Methods Enzymol. 142, 386-397.

Kreps, J.A., and Town, C.D. (1992). lsolation and characterization of a mutant of Arebidopsis thaliane resistant to alpha methyltryptophan. Plant Physiol 99, 269-275.

Kutchan, T.M. (1995). Alkaloid biosynthesis-the basis for metabolic engineering of medicinal plants. Plant Cell 7, 1059-1070.

Lam, H.-M., Coschlgano, K., SchuRz, C, Melo-Oliveira, R., Tjeden, G., Oliveira, I., Ngal, N., Hsleh, M.-H., and Coruui, G. (1995). Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis. Plant Cell 7, 887-898.

Lara, J.C., and Mills, S.E. (1972). Tryptophan synthetase in Euglena gracilis strain G. J. Bacteriol. 110, 1100-1106.

Larsen, P.O. (1981). Glucosinolates. In The Biochemistryof Plants, Vol. 7, €.E. Conn, ed (New York: Academic Press), pp. 501-525.

Last, R.L., and Fink, G.R. (1988). Tryptophan-requiring mutants of the plant Arabidopsis thaliana. Science 240, 305-310.

Last, R.L., Blsslnger, P.H., Mahoney, D.J., Radwanski, E.R., and Ftnk, G.R. (1991). Tryptophan mutants in Arebidopsis: The conse- quences of duplicated tryptophan synthase p genes. Plant Cell3, 345-358.

Lee, H.Y., and Kameya, T. (1991). Selection and characterization of a rice mutant resistant to 5-methyltryptophan. Theor. Appl. Genet. 82, 405-408.

Li., J., Chen, S., Zhu, L., and Last, R.L. (1995a). lsolation of cDNAs encoding the tryptophan pathway enzyme indole-3-glycerol phosphate synthase from Arebidopsis theliene. Plant Physiol. 108, in press.

Li, J., Zhao, J., Rose, A.B., Schmidt, R., and Last, R.L. (1995b). Arabidopsis phosphoribosylanthranilate isomerase: Molecular genetic analysis of triplicate tryptophan pathway genes. Plant Cell 7, 447-461.

Matchett, W.H., and DeMoss, J.A. (1975). The subunit structure of tryptophan synthase from Neuruspore cresse. J. Biol. Chem. 250,

Matem, U. (1994). Dianfhus species (Carnations): In vitro culture and biosynthesis of dianthalexin and other secondary metabolites. In Biotechnology in Agriculture and Forestry, Vol. 28, Y.W. Bajaj, ed (Heidelberg: Springer-Verlag), pp. 170-184.

Mlchalczuk, L., Ribnlcky, D.M., Cooke, T.J., and Cohen, J.D. (1992). Regulation of indole-3-acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiol. 100, 1346-1353.

Mlles, E.W. (1991). Structural basis for catalysis by tryptophan synthase. In Advances in Enzymology and Related Areas of Molecular Biology, Vol. 64, A. Meister, ed (New York: John Wiley and Sons),

Moyed, H.S. (1960). False feedback inhibition: lnhibition of tryptophan biosynthesis by 5-methyltryptophan. J. Biol. Chem. 235,1098-1102.

Nagao, J.T., and Moore, T.C. (1972). Partia1 purification and properties of tryptophan synthase of pea plants. Arch. Biochem. Biophys.

69, 95-101.

2941-2946.

pp. 93-172.

149, 402-413.

934 The Plant Cell

Nlyogl, K.K., and Flnk, G.R. (1992). Two anthranilate synthase genes in Arabidopsis: Defense-related regulation of the tryptophan pathway. Plant Cell 4, 721-733.

Nlyogi, K.K., Last, R.L., Flnk, G.R., and Kelth, B. (1993). Suppres- sors of trp7 fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase p subunit. Plant Cell5,1011-1027.

Nonhebel, H.M., Cooney, T.P., and Slmpson, R. (1993). The route, control and compartmentation of auxin synthesis. Aus. J. Plant Phys- iol. 20, 527-539.

Normanly, J., Cohen, J.D., and Flnk, G.R. (1993). Atabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid. Proc. Natl. Acad. Sci. USA 90,10355-10359.

Normanly, J., Slovin, J.P., and Cohen, J.D. (1995). Rethinking auxin biosynthesis and metabolism. Plant Physiol. 107, 323-329.

Ozenberger, B.A., Brlckman, T.J., and Mclntosh, M.A. (1989). Nucleotide sequence of Escherichia coli isochorismate synthetase gene enC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes. J. Bacteriol. 17l, 775-783.

Poulsen, C., and Verpoorte, R. (1991). Roles of chorismate mutase, isochorismate synthase and anthranilate synthase in plants. Phytochemistry 30, 377-386.

Poulsen, C., Van der Heljden, R., and Verpoorte, R. (1991). Assay of isochorismate synthase from plant cell cultures by high- performance liquid chromatography. Phytochemistry 30,2873-2876.

Poulsen, C., Bongaerts, R.J.M., and Verpoorte, R. (1993). Purifica- tion and characterization of anthranilate synthase from Catharanthus roseus. Eur. J. Biochem. 212, 431-440.

Poulton, J.E., and Moller, B.L. (1993). Glucosinolates. In Methods in Plant Biochemistry, Vol. 9, J.P. Lea, ed (London: Academic Press), pp. 209-237.

Radwanski, E.R., Zhao, J., and Last, R.L. (1995). Atabidopsis fialiana tryptophan synthase alpha: Gene cloning and expression analysis. MOI. Gen. Genet., in press.

Ranch, J.P., Rlck, S., Brotherton, J.E., and Wldholm, J.M. (1983). Expression of 5-methyltryptophan resistance in plants regenerated from resistant cell lines of Datura innoxia. Plant Physiol. 71,136-140.

Rose, A.B., and Last, R.L. (1994). Molecular genetics of amino acid, nucledide and vitamin biosynthesis. In Atabidopsis, E.M. Meyerowitz and C.R. Somerville, eds (Cold Spring Harbor, NY Cold Spring Har- bor Press), pp. 835-879.

Rose, A.B., Casselman, A.L., and Last, R.L. (1992). A phosphoribosylanthranilate transferase gene is defective in blue fluorescent Arabidopsis thaliana tryptophan mutants. Plant Phys- iol. 100, 582-592.

Schurch, A., Miouarl, J., and Hutter, R. (1974). Regulation of tryptophan biosynthesis in Sacchammyces cerevisiae: Mode of action of 5-methyl-tryptophan and 5-methyl-tryptophan-sensitive mutants. J. Bacterioi. 117, 1131-1140.

Singh, B.K., Siehl, D.L., and Connelly, J.A. (1991). Shikimate pathway: Why does it mean so much to so many? In Oxford Surveys of Plant Molecular and Cell Biology, Vol. 7, B.J. Miflin, ed (Oxford: Oxford University Press), pp. 143-185.

Staheli, P., Kradolfer, P., Nlederberger, P., and Hutter, R. (1981). In- hibition of yeast tRNATrp aminoacylation by 4-methyltryptophan. Arch. Microbiol. 129, 146-149.

Takasugi, M., Monde, K., Katsul, N., and Shlrata, A. (1988). Nove1 sulfur-containing phytoalexins from the Chinese cabbage Brassica campestris L. ssp. pekinensis (Cruciferae). Bull. Chem. SOC. Japan

Tam, Y.Y., Slovin, J.P., and Cohen, J.D. (1995). Selection and characterization of a-methyltryptophan resistant lines of Lemna gibba showing a rapid rate of indole-3-acetic acid turnover. Plant Physiol. 107,77-85.

Tsuji, J., Jackson, E.P., Gage, D.A., Hammerschmidt, R., and Somewllle, S.C. (1992). Phytoalexin accumulation in Arabidopsis thdiana during the hypersensitive reaction to Pseudomonas syringae pv. syringae. Plant Physiol. 98, 1304-1309.

Tsuji, J., Zook, M., Hammemchmldt, R., Last, R.L., and Somerville, S.C. (1993). Evidence that tryptophan is not a direct biosynthetic intermediate of camalexin in Arabidopsis thaliana. Physiol. MOI. Plant Pathol. 43, 221-229.

Underhlll, E.W. (1980). Glucosinolates. In Encyclopedia of Plant Phys- iology, Vol. 8, €.A. Bell and B.V. Charlwood, eds (New York: Springer-Verlag), pp. 493-51 1.

Wakasa, K., and Widholm, J.M. (1987). A 5-methyltryptophan resistant rice mutant, MTRl, selected in tissue culture. Theor. Appl. Genet. 74, 49-54.

Widholm, J.M. (1972). Anthranilate synthase from 5-methyltryptophan susceptible and resistant cultured Daucuscamta cells. Biochim. Bic- phys. Acta 279, 48-57.

Wldholm, J.M. (1973). Measurement of the five enzymes which convert chorismate to tryptophan in cultured Daucuscamta cell extracts. Biochim. Biophys. Acta 320, 217-266.

Widholm, J.M. (1976). Selection and characterization of cultured carrot and tobacco cells resistant to lysine, methionine, and proline analogs. Can. J. Bot. 54, 1523-1529.

Wiebe, V.J., and Slpila, P.E.H. (1994). Pharmacology of antineoplas- tic agents in pregnancy. CRC Crit. Rev. Oncol. Hematol. 16,75112.

Wright, A.D., and Neufkr, M.G. (1989). Orange pericarp in maize: Filial expression in a maternal tissue. J. Hered. 80, 229-233.

Wright, A.D., Sampson, M.B., Neuffer, M.G., Mlchalczuk, L., Slovln, J.P., and Cohen, J.D. (1991). Indole-3-acetic acid biosynthesis in the mutant maize orangepericarp, a tryptophan auxotroph. Science

Wright, A.D., Moehlenkamp, C.A., Perrot, G.H.H., Neuffer, M.G., and Cone, K.C. (1992). The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase B. Plant Cell 4, 7ll-7l9.

Yanofsky, C., and Crawkrd, I.P. (1987). The tryptophan operon. In Escherichia coli and Salmonella typhimurium: Cellular and Molecu- lar Biology, Vol. 2, F.C. Neidhardt, J.L. Ingraham, K.B. Low, E. Magasanik, M. Schaechter, and H.E. Umbarger, eds (Washington, DC: American Society for Microbiology), pp. 1453-1472.

Zalkln, H. (1980). Anthranilate synthase: Relationships between bifunctional and monofunctional enzymes. In Multifunctional Proteins, H. Biswanger and E. Schmincke-Ott, eds (New York: John Wiley and Sons), pp. 123-149.

Zalkin, H., and Yanofsky, C. (1982). Yeast gene TRP5: Structure, function, regulation. J. Biol. Chem. 257, 1491-1500.

Zhao, J., and Last, R.L. (1995). lmmunological characterization and chloroplast import of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana. J. Biol. Chem. 270,6081-6087.

61, 285-289.

254, 998-1000.

Documents

tryp biosynthsesis