Plant Physiol. (1996) 110: 51-59

The Arabidopsis thaliana trp5 Mutant Has a Feedback-Resistant Anthranilate Synthase and

Elevated Soluble Tryptophan‘

Jiayang Li2 and Robert L. Last*

Boyce Thompson lnstitute for Plant Research and Section of Genetics and Development, Cornell University, Tower Road, Ithaca, New York 14853-1 801

The first step of tryptophan biosynthesis is catalyzed by anthra- nilate synthase (AS), which is normally subject to feedback inhibi- tion by tryptophan. Three independent frp5 mutants defective in the Arabidopsis fbaliana AS a subunit structural gene ASA7 were iden- tified by selection for resistance to the herbicidal compound 6methylanthranilate. In all three mutants these biochemical changes are caused by a single amino acid substitution from aspar- tate to asparagine at residue position 341. Compared with the enzyme from wild-type plants, the tryptophan concentration caus- ing 50% inhibition of AS activity in the frp5 mutant increased nearly 3-fold, the apparent K,,, for chorismate decreased by approx- imately 50%, and the apparent V,,, increased 60%. As a conse- quence of altered AS kinetic properties, the frp5 mutants accumu- lated 3-fold higher soluble tryptophan than wild-type plants. However, even though the soluble tryptophan levels were increased in frp5 plants, the concentrations of five tryptophan biosynthetic proteins remained unchanged. These data are consistent with the hypothesis that the reaction catalyzed by A. fbaliana AS is rate limiting for the tryptophan pathway and that accumulation of tryp- tophan biosynthetic enzymes is not repressed by a 3-fold excess of end product.

The enzyme AS (EC 4.1.3.27) catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimate) pathway to- ward the biosynthesis of Trp. In addition to its primary role in providing an amino acid for protein synthesis, the Trp biosynthetic pathway of higher plants also produces pre- cursors for synthesis of a variety of important secondary metabolites, including the phytohormone IAA (Normanly et al., 1995), antimicrobial phytoalexins (Tsuji et al., 1993), and other indolic molecules that influence plant-microbe and plant-animal interactions. As a branchpoint enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic second- ary compound biosynthesis (see Fig. 1 for a schematic of the pathway and tvp mutant designations).

’ Supported by grants from the Biotechnology Research Devel- opment Corporation of Peoria, Illinois, the National Institutes of Health (No. GM43134), and a National Science Foundation Presi- dential Young Investigator Award (No. DMB-9058134) to R.L.L.

Present address: Institute of Genetics, Academica Sinica, Bei- jing 100101, Peoples Republic of China.

* Corresponding author; e-mail rll3@comell.edu; fax 1-607-241-1242.

Available information indicates that AS plays a key role in regulation of Trp biosynthesis. In plants, bacteria, and fungi, AS activity is regulated by Trp feedback inhibition (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993). In addition, microbes control the synthesis of Trp biosynthetic enzymes in response to amino acid starvation. For example, Esckerickia coli regulates the expression of the trp operon through transcriptional repression and attenu- ation (Yanofsky and Crawford, 1987), whereas fungi dere- press the transcription of genes encoding Trp biosynthetic enzymes in response to starvation for various amino acids (Hinnebusch, 1992).

In contrast to the wealth of knowledge about the regu- lation of microbial amino acid biosynthesis, information concerning the genetic a n d biochemical regulation of the plant Trp pathway is just beginning to emerge (Radwanski and Last, 1995). For example, AS mRNA levels increase in response to bacterial pathogen infection and wounding in A. tkaliana (Niyogi and Fink, 1992; Niyogi et al., 1993) and funga1 elicitor treatment in Ruta gvaveolens (Bohlmann et al., 1995). This increased mRNA accumulation presumably allows enhanced synthesis of secondary products, such as indolic phytoalexins (Tsuji et al., 1992) in A. thaliana and anthranilate-derived acridone alkaloids in R. graveolens. Altered regulation of AS is also observed in Trp biosyn- thetic mutants (Fig. 1): the activity of the enzyme is higher in tvpl-100 (Niyogi et al., 1993) and tvp2-2 (Last et al., 1991), and the concentration of AS a subunit is increased in a11 13 trp2 mutants tested (Barczak et al., 1995). Unlike in bacteria, the steady-state leve1 of AS a subunit mRNA did not change in A. tkaliana in response to growth of plants on 50 PM Trp (Niyogi and Fink, 19921, suggesting that accu- mulation of Trp biosynthetic enzymes is not repressed by end product.

Two distinct subunits, a and /3 (also called components I and 11, respectively), are required for Gln-dependent AS activity. The a subunit binds to chorismate and catalyzes its aromatization, and the /3 subunit transfers an amino group from Gln. Biochemical studies of the AS complex from bacteria and fungi revealed that the Trp feedback inhibition of AS results from the binding of one molecule of

Abbreviations: AS, anthranilate synthase; Col-O, Columbia ecotype of A. thaliana; Ler, Landsberg erecta line of A. thaliana; MA, methylanthranilate; MT, methyltryptophan; PNS, plant nutrient medium with 0.5% SUC.

51

52 L i and Last Plant Physiol. Vol. 11 0, 1996

Phenylalanine

Tyrosine + e+---- Chorismate

t r ~ 5 I ASa

6MA

4 i 4 4 I

4MT

C O .- .- c e c c Y o m m U 8

.-

d

Anthranilate trpl i PAT

PRA

i CDRP

lnGP trp3 4 TSa

lndole trp2 I TsO

4 InGPS

Figure 1. Proposed mechanism for toxicity of 6MA. 6MA is proposed to exert its toxic effects indirectly by conversion into 4MT. In this scheme, 6MA resistance can be mediated by reduced activity of the enzymes that convert anthranilate to Trp (recessive t rp l , trp2, and trp3 alleles) or by mutations that render AS less sensitive to inhibition by Trp or Trp analogs. The available mutants of the A. thaliana Trp biosynthetic pathway are given at the corresponding steps (trpl-trp4). AS a, AS (EC 4.1.3.27) a subunit; ASP, AS P subunit; CDRP, 1-(0- carboxyphenylami no)-1 -deoxyri bu lose-5-phosphate; I nC P, indole- 3-glycerol phosphate; InCPS, lnGP synthase (EC 4.1.1.48); PAI, phosphoribosylanthranilate isomerase; PAT, phosphoribosylanthra- nilate transferase (EC 2.4.2.1 8); PRA, phosphoribosylanthranilate; TSa, Trp synthase (EC 4.2.1.20) a subunit; TSP, Trp synthase p subunit. Mutants t rp l , trp2, trp3, and trp4 are defective in PAT, TSP, TSa, and ASP, respectively.

Trp to the a subunit (Henderson and Zalkin, 1971). The microbial AS enzymes can be desensitized to allosteric regulation by mutations in this subunit. DNA sequencing and comparison of microbial wild type and their feedback- resistant mutants have identified two conserved domains of the AS a subunit involved in feedback regulation (Mat- sui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993).

Various toxic compounds have been used to isolate Trp biosynthetic pathway mutants in plants. For example, 5MA was successfully used to isolate plant auxotrophic mutants defective in three different genes, t r p l , trp2, and trp3 (Last and Fink, 1988; Last et al., 1991; E.R. Radwanski, A.J. Barczak, and R.L. Last, unpublished results), and mutants of Cklamydomonas reinkardtii (Dutcher et al., 1992). An al- lelic series of trp2 mutants with defects in the Trp synthase /3 subunit was identified using 5-fluoroindole selection (Barczak et al., 1995). Mutants resistant to 5MT or aMT were reported in A. tkaliana (Koornneef and van Loenen Martinet, 1983; Kreps and Town, 1992), maize (Kang and Kameya, 1993), Lemna gibba (Tam et al., 1995), and Oryza satiua (Lee and Kameya, 1991). However, the efficiency and

specificity of selection with these analogs have not been systematically investigated. Although Trp analog-insensi- tive mutants were isolated from the plants A. tkaliana (Kreps and Town, 1992), Lemna gibba (Tam et al., 1995), and Nicotiana tabacum (Widholm, 1972a, 1972b), the nature of the amino acid changes resulting in altered allosteric reg- ulation of plant AS was not reported, and it is not known whether these are caused by AS structural gene mutations.

In this paper, we report the isolation of A. tkaliana Trp biosynthetic pathway mutants using 6MA. Among this collection three semidominant mutants were characterized and shown to contain elevated soluble Trp due to altered feedback regulation resulting from a single amino acid residue substitution in the AS a subunit encoded by A S A 1 . The dominance of the trp5 mutation suggests that this allele could be used to increase free Trp levels in transgenic plants, thereby improving the nutritional quality. This dominant mutation may also serve as a selectable marker in transformation experiments.

MATERIALS A N D METHODS

Plant Crowth

Arabidopsis tkaliana mutants and parenta1 Col-0 wild-type plants were grown on sterile agar medium or Redi-Earth soil-less mixture (Grace-Sierra, Milpitas, CA) at 22°C under continuous illumination of 80 to 120 pmol m-’ s-l PAR from mixed cool-white and incandescent lights or from MVR-400/U multivapor metal halide bulbs (General Elec- tric) as described by Last and Fink (1988). For all biochem- ical assays, tissues of the plants grown in a soil-less mixture were harvested freshly for extraction. Trp auxotrophy was assayed by comparing growth under 180 pmol m-’ s-’ PAR of seeds on minimal PNS medium containing or lack- ing Trp (Last et al., 1991). Analogs of anthranilate and Trp were dissolved in neat DMSO or a 1:l mixture of DMSO and sterile water.

Mutant Selection

Trp biosynthetic pathway mutants were selected by growing M, seeds on 25 mL of sterile PNS agar medium containing 300 p~ 6MA and 25 p~ L-Trp at a density of 500 seeds per 100-mm-diameter Petri plate under continuous illumination (80-120 pmol m-’ s-’ PAR) at 22°C. M, seeds were obtained by mutagenesis with N-nitroso-N-methyl- urea or y irradiation as described by Barczak et al. (1995). Putative resistant plants were identified after 2 to 4 weeks of growth, transferred to PNS plates containing 50 p~ Trp for 1 week to recover, and then transplanted to a soil-less mixture to self-pollinate. The 6MA-resistance phenotype of M,, F,, or F, seeds was retested on PNS plates containing 6MA and Trp.

AS Activity Assay

Extracts for the assay of AS activity were prepared as previously described (Last and Fink, 1988). The Gln-depen- dent AS activity was assayed in a 500-pL reaction mixture containing 35 mM Tris-HC1, pH 8.0, 50 PM chorismate, 20

Arabidopsis Anthranilate Synthase Feedback Resistance 5 3

mM L-Gln, 2.0 mM MgCl,, 50 p~ EDTA, and 50 p~ DTT. The reaction was started by the addition of 25 pL of plant extract to the reaction mixture, incubated at 30°C for 15 min, and terminated by the addition of 50 pL of 1.0 N HCl. The anthranilate produced was extracted with 1.2 mL of ethylacetate and centrifugated at 16,0008 for 3 min in a microcentrifuge at room temperature. The anthranilate in the upper phase was then quantified with a Perkin-Elmer MPF-44B fluorescence spectrophotometer (excitation at 340 nm and emission at 400 nm). For AS kinetic studies, the concentrations of chorismate or L-Trp were varied as indi- cated. In preliminary experiments it was shown that the production of anthranilate was linear with respect to time during a 45-min assay period at the chorismate concentra- tions used (J. Li, unpublished data), indicating that the reactions are in substrate excess throughout the 15-min assay period.

Free Trp Measurement

To determine the content of soluble Trp in Arabidop- sis, 50 mg of freshly harvested plant material were ho- mogenized with 200 pL of water containing 13 mg of polyvinylpolypyrrolidone, and the suspension was cleared by centrifugation at 16,OOOg for 5 min in a mi- crocentrifuge at room temperature. The supernatant was then transferred into a new tube, and 200 pL of methanol were added. After incubating for 5 min at room temper- ature, the mixture was centrifuged for 5 min and the supernatant was used for Trp measurement as described previously (Heinrikson and Meredith, 1984). Extraction with polyvinylpolypyrrolidone allowed reliable Trp quantitation, because this treatment reduced the amounts of other UV absorptive compounds that eluted near the Trp HPLC peak. However, this technique did not give quantitative extraction of more polar amino acids. To measure the other amino acids, 50 mg of plant tissue were homogenized with 200 pL of extraction so- lution (20% ethanol and 0.1 N HC1). The suspension was centrifuged at 16,0008 for 5 min twice, and the superna- tant was used for soluble amino acid analysis.

Genetic Analysis of the trp5 Mutants

Standard genetic analysis was performed as previously described (Last and Fink, 1988), and resistance was assayed after 3 to 4 weeks of growth on 300 p~ 6MA. An F,- mapping population was generated from crosses between each of the independently isolated trp5 mutants (in the Col-O genetic background) and the wild-type Ler ecotype. The F, progeny plants were allowed to self-pollinate, and F, plants were scored for 6MA resistance. Genomic DNA was prepared as described before (Bernatzky and Tanks- ley, 1986) from the parenta1 plants, the F,, and 55 strongly resistant trp5/trp5 F, progeny plants from each cross. The genetic linkage between the trp5 mutation and known polymorphic DNA markers was tested using the co-dom- inant amplified polymorphism sequence PCR method (Konieczny and Ausubel, 1993).

Cloning and Sequencing ASA 1

ASA2 genomic DNA was amplified from the LIAl, LIA2, and LIA3 lines and the control Col-O wild-type plants were amplified by PCR using primers JLlOl and JL102 (Table I) at an annealing temperature of 58°C. The 3.7-kb PCR prod- ucts covered the complete ASA2 genomic sequence. Two independent, PCR-amplified ASA2 products from the wild type and each of the mutant lines were directly cloned into pCRII plasmid (Invitrogen, San Diego, CA). Single- stranded DNA from the eight templates was prepared as described previously (Li et al., 19951, and DNA sequencing was performed using a DNA-sequencing kit (Amersham) with primers designed based on the published ASA2 se- quence (Niyogi and Fink, 1992), as given in Table I, as well as with the forward and reverse primers described by Niyogi and Fink (1992). Because PCR amplification with Tuq polymerase is mutagenic, we only report nucleotide changes between wild type and the trp5 mutant sequences that were found in both clones from each pair of indepen- dent PCR reactions.

lmmunoblot Analysis

Production of rabbit antisera, protein gel electrophoresis, immunoblotting, and radioactivity quantitation with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) were previously described (Zhao and Last, 1995). Protein concentration was measured with a protein dye-binding assay kit (Bio-Rad), using lysozyme as the standard.

RESULTS

lsolation of 6MA-Resistant Mutants

Previous work from this laboratory indicated that an- thranilate analogs can be used to select for loss-of-function mutants in the A. tkaliana Trp biosynthetic pathway (Last and Fink, 1988; Li et al., 1995). Although selection for 5MA resistance identified recessive mutations in three comple- mentation groups (Last and Fink, 1988; Last et al., 1991; E.R. Radwanski and R.L. Last, unpublished data), it did not provide an absolute selection for Trp biosynthetic mutants. Only one-half of the putative 5MA-resistant M, plants produced resistant progeny, whereas the remainder were “escapers.” This made it difficult to collect a large number

Table 1. Sequences of the primers used in this studv Name

JL1 O1 JL102 JL1 03 JL104 JL105 JL106 JL107 JL l08 JL1 O9 JL110 JLI 11 ]LI 1 2

Sequence

5’-GAGAAAGTCGACCGTTCGTATCGTTTGC-3 ’ 5‘-CAATCCATCCTCAACTCGCTATTTGCCG-3’

5’-TTACTCATTTAGCCGTCACCTCTG-3 ‘ 5‘-GGTTGAACGACATTTATGTCAAGA-3’ 5 ’-CCTTTTTATCTCTTAATATGTCTC-3’

5’-CTCCTTTATCTTGTTCATGAAGTC-3’ 5’-ACTGAGACATCCATCTTAGTTTCC-3 ‘ 5 ’-GCCTACTATTCTTAAAAGTTGTTC-3 ’ 5’-CAACTTTGCATTTTCTACTTGTGG-3 ‘ 5’-GCATTAATCTTTATATCATTTTTG-3‘ 5 '-GTAG ATCAAATATCTAAAGTCTCG-3 ’ 5’-CCCTTTTCCTGCACTCTCCTTCAC-3’

54 Li and Last Plant Physiol. Vol. 110, 1996

of mutants in the Trp pathway. In an attempt to find a moreeffective selection procedure, we tested the relative toxicityof four other commercially available anthranilate analogs.Figure 2A shows that, of the five analogs tested, 6MA is themost potent inhibitor of the growth of wild-type Col-0. Incontrast, growth of wild-type plants was not inhibited bythe 3-methyl and 6-fluoro analogs of anthranilate. Giventhe 6MA resistance of the trpl-100 mutant (which has adefect in the phosphoribosylanthranilate transferase), it isplausible that the strong toxicity of 6MA is due to biosyn-thetic conversion to 4MT (Fig. 1), which is toxic to wild-type A. thaliana (Fig. 2B). This suggested that 6MA couldyield both loss-of-function mutants and regulatory mutantswith increased Trp biosynthesis.

Trp biosynthetic pathway mutants were sought by selec-tion for 6MA resistance. Eighty independent putative mu-tants resistant to 6MA were isolated out of approximately300,000 M2 seeds. These plants produced green true leaves onthe selection medium, whereas growth of the wild-typeplants was completely inhibited, produced chlorotic cotyle-dons, and eventually died, as shown in Figure 2A (compareCol-0 to trpl-100 or trp5 mutants). Among these putativeresistant mutants, most were subsequently identified as alle-les of trpl, trp2, or trp3, based on genetic crosses and immu-noblot analysis of Trp biosynthetic pathway protein levels(data not shown). However, results from a combination ofphenotypic, genetic, and biochemical analyses indicated thatthree of the 6MA-resistant lines (LIA1, LIA2, and LIAS) were

6MA 5MA 5FA 3MA 6FA DMSO

B

Figure 2. Effects of anthranilate and Trp analogs on wild-type and mutant A. thaliana plants. LIA1, LIA2, and LIA3, threeindependent lines containing the frp5-7 mutation; trpl-100, a mutant defective in phosphoribosylanthranilate transferase(Rose et al., 1992). A, Plants grown for 2 weeks following seed imbibition on PNS agar containing the indicated anthranilateanalogs (300 JJ.M). FA, Fluoroanthranilate; DMSO, 4.5 mM DMSO control. B, Plants grown for 2 weeks on PNS mediumcontaining the indicated Trp analogs (50 JUM). Water, No inhibitor control. The three arrows indicate presumed in vivoconversion of anthranilate analogs into the corresponding Trp analogs.

Arabidopsis Anthranilate Synthase Feedback Resistance 55

AS mutants with altered Trp allosteric regulation. As docu- mented below, these independently isolated mutants contain the identical mutation, which is designated trp5-1. The mu- tagen for LIA1 and LIA2 was N-nitroso-N-methylurea, and LIA3 was induced by y irradiation.

Other trp5 Mutant Phenotypes

Phenotypic analysis of the trp5 mutants indicated the possibility that the biochemical defect was distinct from other A. thaliana mutants isolated as resistant to anthrani- late analogs. In contrast to previously characterized reces- sive loss-of-function mutations in biosynthetic pathway enzymes such as phosphoribosylanthranilate transferase (i.e. trp1-100), trp5 plants are strongly cross-resistant to 4MT, 5MT, 7MT, and aMT (Fig. 28). This suggests that the trp5 mutant may accumulate increased Trp pools or have a defect in uptake of Trp analogs, rather than a block in Trp biosynthesis. Consistent with this hypothesis, the trp5 plants are prototrophic under sterile culture conditions that cause a Trp requirement in characterized auxotrophic mutants (data not shown). The only developmental differ- ence noted was that the trp5 mutant plants growing in a soil-less mixture produce a flower stalk and mature flowers approximately 3 d earlier than the control wild-type plants (data not shown).

Cenetic Characterization of trp5 Mutants

To explore whether the 6MA-resistance phenotype was conferred by a single Mendelian trait, crosses were made between these mutants and the Ler wild type, and the resultant F, progeny were allowed to self-pollinate to pro- duce a segregating F, population (Table 11). Progeny seeds were plated on 6MA-containing medium and their pheno- types scored between 3 and 4 weeks after planting. As presented in Table 11, a11 of the F, progeny showed mod- erate resistance to the selection agent, suggesting the pos- sibility of a semidominant resistance trait. Consistent with this hypothesis, the F, population contained three distinct classes of progeny in a 1:2:1 ratio (strongly resistant:mod- erately resistant:wild-type sensitivity). Analysis of F, plants from eight strongly resistant F, progeny from each

Table [I. Genetic analysis of the trp5 mutants

The crosses were made between wild-type Ler (female parent) and homozygous trp5 plants (polien donor). The trp5 plants were F, individuals derived from a single backcross of the original lines with the COLO wild-type parent. L lAl , LIA2, and LIA3 are the designations of the original 6MA-resistant mutant lines, each of which contains the trp5-7 mutation.

Resistance to 6MA

Sensitive Cross Progeny Strong Moderate resistance resistance

Wild type X LIA1 F, O 28 O

Wi ld type X LIA2 F, O 54 O F2 112 224 112

Wi ld type X LIA3 F, O 42 O

F2 150 282 151

F2 142 296 136

cross confirmed that the strongly resistant F, plants were homozygous for the 6MA-resistance alleles, because a11 F, progeny were strongly resistant.

The observation that the three independently isolated mutants exhibited similar inhibitor resistance and were semidominant suggested the possibility that the three lines harbored mutations in the same gene. Because it is known that mutations of the AS a subunit in microbes can lead to reduced feedback inhibition by Trp, it seemed plausible that trp5 mutations might affect feedback regulation of the committing enzyme in the Trp biosynthetic pathway, AS. Although there are two AS a structural genes in A. thaliana (ASA1 and ASA2), ASA2 is the more highly expressed locus (Niyogi and Fink, 1992). Considering these facts, we tested the hypothesis that the analog resistance was conferred by mutations in ASAl by asking whether trp5 was genetically linked to ASAZ.

To locate the mutations on the A. thaliana genome, F, mapping populations were generated from crosses be- tween the TRP5+ wild-type Ler and homozygous 6MA- resistant, Col-O-derived mutants. Genomic DNA was pre- pared from 55 individuals that showed strong resistance to 6MA (and were presumed homozygous for the resistance allele) in each F, mapping population, and the co-dominant amplified polymorphism PCR-based genetic mapping technique (Konieczny and Ausubel, 1993) was used to test linkage between the mutations and characterized restric- tion fragment length polymorphisms. Consistent with the hypothesis that the tup5 mutations were in ASA1, 6MA resistance and the ASA1 gene showed absolute genetic linkage in a population of 165 homozygous F, progeny (55 highly resistant plants per mutant). As controls it was demonstrated that severa1 markers on other chromosomes segregated independently of the 6MA-resistance locus (e.g. the ADH locus on chromosome 1 showed 8 Col-O homozy- gotes, 9 Ler homozygotes, and 28 heterozygotes). These data suggest that the mutations in lines LIA1, LIA2, and LIA3 are alleles of the ASA2 gene on chromosome 5.

A Single Amino Acid Change in ASA1 of the trp5 Mutants

To determine the nature and location of the mutation in the three trp5 mutants, clones of two independent PCR products covering the complete ASA1 genomic DNA re- gions from LIA1, LlA2, LIA3, and wild-type plants were sequenced. Surprisingly, each trp5 sequence had the same G-to-A base transition, resulting in an aspartate to Asn amino acid substitution at position 341 of the A. thaliana AS a protein (Fig. 3). To our knowledge, mutations in this region of the AS a protein have not been reported to result in feedback insensitivity in microbial AS (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993). How- ever, it is located adjacent to one of the two domains that contain amino acids mutable to feedback resistance in mi- crobes (these sites are represented by asterisks in Fig. 3).

AS Kinetic Study

If the ASAl structural gene mutation identified in trp5 lines caused the observed resistance to growth inhibition by the

56 L i and Last Plant Physiol. Vol. 11 O, 1996

1 111 338 595

WT FLFESVEP(X)i@VV TFADPFEVYRALRVVNPSPYMGYLQARGCILVASS

trp5 4 N

Figure 3. The location of the amino acid substitution in the trp5 mutants. The numbers on the top indicate the amino acid positions on the protein encoded by ASA7 (Niyogi and Fink, 1992). The asterisks indicate the positions of amino acids that can strongly affect the Trp inhibition of microbial AS (Matsui et al., 1987; Caligiuri and Bauerle, 1991 ; Graf et al., 1993). The arrow shows the substitution of Asp to Asn at amino acid 341 in trp5 mutants. WT, Wild type.

anthranilate and Trp analogs, the AS enzyme activity would be expected to be altered. To determine whether the mutant AS was altered in its sensitivity to Trp feedback inhibition, both mutant and wild-type AS enzyme activities were mea- sured in the presence of increasing concentrations of L-TT. As shown in Figure 4B, the tup5 mutant enzyme is indeed more resistant to Trp inhibition: the Trp concentrations caus- ing 50% AS activity inhibition (apparent K, for L-Trp) were 3 p~ for the wild type and 8 ~ L M for LIA2, a 2.5-fold increase. The kinetic parameters of AS activity from wild-type and trp5 mutant plants were determined from the Lineweaver-Burk plots shown in Figure 4A. The apparent K , for chorismate was 10 p~ for the tup5 AS compared with 21 p~ for the wild-type enzyme, a 50% decrease. The apparent V,,, of the mutant AS was 60% higher than the wild-type enzyme. The kinetic data for the tup5 mutant and wild-type plants are summarized in Table 111.

Elevated Soluble Trp in trp5 Mutant Plants

The observed changes in the AS kinetic parameters of the tup5 mutant, and Trp analog resistance, suggested that the free Trp content might be elevated in the mutant plants. Figure 5 shows that the free Trp leve1 in tup5 was elevated approximately 3-fold compared with the wild type. This tu@-elevated Trp concentration was consistent in a11 sam- ples tested, despite the observation that soluble Trp levels expressed on a fresh tissue weight basis varied among the organs analyzed (being especially high in reproductive tissue) in both wild type and trp5-2 (line LIA2). A11 other amino acids were found to vary 530% when comparing wild-type and mutant plants at both 14 and 28 d postim- bibition (data not shown).

N o Repression of Trp Pathway Cenes by Elevated, Endogenous Soluble Trp

The availability of plants with elevated Trp permitted an evaluation of whether A. thaliana down-regulates the accumu- lation of Trp biosynthetic enzymes in response to amino acid accumulation. Immunoblot analysis of the three independent isolates of tup5-2 and wild-type control plants demonstrated that there were no significant changes in accumulation of cross-reactive proteins identified by antibodies to AS a, phos- phoribosylanthranilate transferase, phosphoribosylanthrani-

late isomerase, or Trp synthase a and p subunits (Table 111). These results indicate that a 3-fold increase of Trp pools does not reduce the steady-state concentrations of Trp biosynthetic enzymes in A. thaliana.

DISCUSSION

In this study we have described the isolation of three independent A. thaliana AS a subunit structural gene mu- tants with altered enzymatic properties. Although these mutants were originally identified as resistant to 6MA, they are also able to grow on concentrations of Trp analogs that are inhibitory to wild type. As expected for a Trp analog-resistance AS mutation, tup5 is semidominant, causes elevated accumulation of soluble Trp, and produces

A “‘“3 - 8 . E, o 0.31

7

/ - 8 o 0.3- . E, 7 - a, - 1 .- 0.2-

s 5 c i5 0.1 1

A

- a, - 1 .- 0.2-

s 5 c i5 0.1 1

A

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

1 /[chorismate], 1 /pM

O 20 40 60 80 100

[L-tryptophan], pM

Figure 4. Kinetic analysis of AS of wild-type and feedback-resistant trp5 mutant line LIA2. A, Lineweaver-Burk plots of AS activity of extracts from 4-week-old wild-type (wt) and trp5 mutant rosette leaves. Twenty- five microliters of protein extract containing 60 pg of total protein were used in each assay. B, Relative activities of AS from 4-week-old wild- type (wt) and mutant rosette leaves, measured as described in ”Materials and Methods” with addition of L-Trp to 0.0, 1.0, 2.5, 5.0, 10.0, 20.0, 50.0, or 1 O0 p ~ . The Trp concentrations yielding 50% inhibition of AS activities (apparent K, for L-Trp) for wild-type and trp5 plants are ap- proximately 3 and 8 p ~ , respectively.

Arabidopsis Anthranilate Synthase Feedback Resistance 57

Table 111. Summary of characteristics of trp5-I, trpl-100, and wild-type plants

S, Sensitive; R, resistant.

Characteristic Wild Type trp5-l” trpl-700

Phenotype Resistance to 6MA Resistance to 4MT Resistance to 5-fluoroindole

Apparent K,,, for chorismate

Apparent V,,, (nmol min-’

Apparent K, for L-Trp (FM) Free Trp leve1 (nmol g-’ fresh

Kinetic properties

(FM)

[mg proteinl-’)

tissue) 14-d-old rosette leaf 28-d-old rosette leaf Buds/flowers Young siliques

Relative abundance of the path way enzymes

AS a Phosphoribosylanthranilate

transferase Phosphoribosylanthranilate

isomerase Trp synthase a Trp synthase

due 341 AS (Y subunit amino acid resi-

S S S

21

74

3

18 15 81 27

1 O0 1 O0

1 O0

1 O0 1 O0 ASP

R R R

10

120

8

54 40

31 O 79

1 O 0 110

110

80 110 Asn

R S S

a The line LIA2 was used as the trp5-7 mutant.

a catalytically altered enzyme activity. This mutant enzyme not only has a reduced sensitivity to Trp inhibition (in- creased apparent Ki) but appears to be more catalytically active, with an increased apparent V,,, and decreased apparent K , for chorismate. A11 of these properties are due to a single missense mutation in ASA2 (Fig. 3).

Although extensive analyses of microorganisms have revealed various AS a mutations that alter allosteric regu- lation (Matsui et al., 1987; Caligiuri and Bauerle, 1991; Graf et al., 1993), the trp5 mutation alters an amino acid at a position not previously reported to have been mutated. In contrast to the diversity of sites mutable to feedback resis- tance in microbes, the three independently isolated A. thali- una alleles (in lines LIA1, LIA2, and LIA3) identified from two different mutagen treatments a11 have the same muta- tion. It was recently demonstrated that the A. tkaliana amt-1 mutation, which was identified by selection for resistance to a-methyltryptophan (Kreps and Town, 1992), has the same mutation as trp5-2 (Kreps et al., 1996). Taken to- gether, these results suggest that aspartate-341 is important for the normal regulation of A. thaliana AS or that this region of the gene is especially easily mutable. Perhaps the change from a negatively charged to a neutra1 amino acid residue alters the enzyme conformation, thereby resulting in a higher affinity for chorismate (the decrease of apparent K,, Table 111) and the desensitization to Trp feedback in- hibition (the increase of apparent Ki, Table 111).

Despite the repeated isolation of trp5-2, it is unlikely that aspartate-341 is the only amino acid in A. tkaliana ASA that can be mutated to give strong resistance to Trp analogs. For example, ASA1 and ASA2 mutants that contain a Ser-to- Phe change at residue 115 of the Asa1 protein sequence (Niyogi and Fink, 1992) were constructed by in vitro mu- tagenesis and expressed as cDNAs driven by the cauli- flower mosaic virus 355 promoter in transgenic A. thaliana (Niyogi, 1993). These changes, which are analogous to a dominant feedback insensitivity mutation characterized in Salmonella typhimurium t rpE (Caligiuri and Bauerle, 1991), led to plants that were moderately resistant to 5MT. It is plausible that this and other mutations would be identified in an exhaustive selection for Trp analog-resistant mutants.

Analogs of intermediates in the Trp biosynthetic path- way were previously used for isolation of mutants in the flowering plant A. thaliana (Last and Fink, 1988; Barczak et al., 1995) and the alga C. reinkardtii (Dutcher et al., 1992). This systematic comparison of the toxicity of sev- era1 commercially available analogs revealed significant differences in the efficacy of these compounds for dis- tinguishing between Trp pathway mutants and wild- type A. thaliana (Fig. 2A). These studies indicated that 6MA is the most effective analog tested, consistent with its known herbicidal activity (Thomas, 1984). The agree- ment between the relative toxicity of three anthranilate analogs (6MA > 5MA > 3MA) and their corresponding Trp analogs (4MT > 5MT > 7MT) suggests that toxicity is due to metabolism of the anthranilate compounds (compare the wild-type plant phenotypes in Fig. 2, A and B, that are connected by arrows). This argument is strengthened by the observation that the loss-of-function pathway mutants are highly resistant to anthranilate analogs but not to Trp analogs. Taken together, our results indicate that 4MT should be an effective agent for selection of mutants with increased Trp biosynthesis.

400

T

leaf leaf bud/flower silique 14d 28d

Figure 5. Soluble Trp content of wild-type and trp5 mutant line LIA2. Mean values and SES are reported for soluble Trp levels of 14- or 28-d-old rosette leaves (seven and five independent samples, respec- tively) and duplicate samples each of buds/flowers and young green siliques (seed pods). W, Wild-type values; B, trp5 values.

58 Li and Last Plant Physiol. Vol. 11 O, 1996

The availability of Trp-overproducing plants permitted a test of the hypothesis that plants might down-regulate this pathway in response to excess end product accumulation. Quantitative immunoblot experiments indicate that there is no change in accumulation of five biosynthetic proteins in response to 3-fold higher Trp accumulation (Table 111). This result is significant in two regards. First, it indicates that AS catalyzes the rate-limiting step in this pathway. The change of enzymatic characteristics of AS alone causes Trp overproduc- tion, whereas the concentrations of pathway enzymes are not affected. Second, it extends published results indicating that addition of exogenous Trp to sterile growth medium does not affect the accumulation of AS a RNA in wild-type plants (Niyogi and Fink, 1992). Taken together, these results suggest that accumulation of excess free Trp does not repress the expression of the Trp biosynthetic pathway enzymes in plants. In contrast, loss-of-function trp2 mutants have in- creased accumulation of AS a protein, suggesting that the plant does respond to amino acid privation.

The availability of a cloned, dominant AS mutation that causes elevated soluble Trp may have utility in agriculture. Trp is a nutritionally limiting amino acid in plants such as maize (Glover and Mertz, 1987). Furthermore, there are a variety of medically important secondary metabolites derived from the Trp pathway (Kutchan, 1995). Production of trans- genic plants expressing a dominant feedback-resistance trp5 allele may lead to increased nutritional value of crop plants or permit increased synthesis of useful secondary products. Fi- nally, the observation that plants heterozygous for trp5 are resistant to toxic anthranilate and Trp analogs suggests that this may be a useful selectable marker for plant transforma- tion experiments.

ACKNOWLEDCMENTS

We thank Melissa Ho for technical assistance, Katherine Denby and Alan Rose for comments concerning the manuscript, and the Biotechnology Center of Cornell University (Ithaca, NY) for mea- surement of free Trp.

Received August 25, 1995; accepted October 5, 1995. Copyright Clearance Center: 0032-0889/96/110/0051/09.

LITERATURE ClTED

Barczak AJ, Zhao J, Pruitt KD, Last RL (1995) 5-Fluoroindole resistance identifies tryptophan synthase beta subunit mutants in Arabidopsis thaliana. Genetics 140 303-313

Bernatzky R, Tanksley SD (1986) Toward a saturated linkage map in tomato based on isozymes and random complementary DNA sequences. Genetics 112 887-898

Bohlmann J, DeLuca V, Eilert U, Martin W (1995) Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes. Plant J 7: 491-501

Caligiuri MG, Bauerle R (1991) Identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. Evidence for an amino-terminal regulatory site. J Biol Chem 266: 8328-8335

Dutcher SK, Galloway RE, Barclay WR, Poortinga G (1992) Tryp- tophan analog resistance mutations in Chlamydomonas rein- hardtii. Genetics 131: 593-607

Glover DV, Mertz ET (1987) Corn. In RA Olson, KJ Frey, eds, Nutritional Quality of Cereral Grains: Genetic and Agronomic

Improvement. Soil Science Society of America, Madison WI, pp

Graf R, Mehmann B, Braus GH (1993) Analysis of feedback- resistant anthranilate synthases from Saccharomyces cerevisiae. J Bacteriol 175: 1061-1068

Heinrikson RL, Meredith SC (1984) Amino acid analysis by re- verse-phase high-performance liquid chromatography: precol- umn derivatization with phenylisothiocyanate. Ana1 Biochem

Henderson EJ, Zalkin H (1971) On the composition of anthranilate synthase-anthranilate 5-phosphoribosylpyrophosphate phos- phoribosyltransferase from Salmonella typhimurium. J Biol Chem

Hinnebusch AG (1992) General and pathway-specific regula- tory mechanisms controlling the synthesis of amino acid bio- synthetic enzymes in Saccharomyces cerevisiae. In EW Jones, JR Pringle, JB Broach, eds, The Molecular and Cellular Biology of the Yeast Saccharomyces, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 319-414

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

Konieczny A, Ausubel FM (1993) A procedure for mapping Ara- bidopsis mutations using co-dominant ecotype-specific PCR- based markers. Plant J 4: 403410

Koornneef M, van Loenen Martinet EP (1983) The isolation of a 5-methyltryptophan insensitive mutant by selection at the seed- ling stage. Arabidopsis Inf Serv 20: 104-108

Kreps JA, Tilak T, Dong W, Town CD (1996) Molecular basis of a-methyltryptophan resistance in amt-2, a mutant of Arabidopsis thaliana with altered tryptophan metabolism. Plant Physiol (in press)

Kreps JA, Town CD (1992) Isolation and characterization of a mutant of Arabidopsis thnlinna resistant to alpha methyltrypto- phan. Plant Physiol 99: 269-275

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

Last RL, Bissinger PH, Mahoney DJ, Radwanski ER, Fink GR (1991) Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase

Last RL, Fink GR (1988) Tryptophan-requiring mutants of the plant Arabidopsis thaliana. Science 240 305-310

Lee HY, Kameya T (1991) Seledion and characterization of a rice mutant resistant to 5-methyltryptophan. Theor Appl Genet 8 2 405-408

Li J, Zhao J, Rose AB, Schmidt R, Last RL (1995) Arabidopsis phosphoribosylanthranilate isomerase: molecular genetic analysis of triplicate tryptophan pathway genes. Plant Cell 7:

Matsui K, Miwa K, Sano K (1987) Two single base pair substi- tutions causing desensitization to tryptophan feedback inhi- bition of anthranilate synthase and enhanced expression of tryptophan genes of Brevibacterium lactofermentum. J Bacteriol

Niyogi KK (1993) Molecular and genetic analysis of anthranilate synthase in Arabidopsis thaliana. PhD dissertation. Massachusetts Institute of Technology, Cambridge, MA

Niyogi KK, Fink GR (1992) Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan path- way. Plant Cell 4 721-733

Niyogi KK, Last RL, Fink GR, Keith B (1993) Suppressors of trpl fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit. Plant Cell 5 1011-1027

Normanly J, Slovin JP, Cohen JD (1995) Rethinking auxin biosyn- thesis and metabolism. Plant Physiol 107: 323-329

Radwanski ER, Last RL (1995) Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. Plant Cell7:

Rose AB, Casselman AL, Last RL (1992) A phosphoribosylanthrani- late transferase gene is defective in blue fluorescent Arabidopsis thaliana tryptophan mutants. Plant Physiol 100 582-592

Tam YY, Slovin JP, Cohen JD (1995) Selection and characteriza- tion of a-methyltryptophan resistant lines of Lemna gibba show-

183-336

136 65-74

246: 6891-6898

69: 95-101

genes. Plant Cell3: 345-358

447-461

169: 5330-5332

921-934

Arabidopsis Anthrani late Synthase Feedback Resistance 59

ing a rapid rate of indole-3-acetic acid turnover. Plant Physiol

Thomas G J (1984) Herbicidal activity of 6-methylanthranilic acid and analogues. J Agric Food Chem 3 2 747-749

Tsuji J, Jackson EP, Gage DA, Hammerschmidt R, Somerville SC (1992) Phytoalexin accumulation in Arabidopsis thaliana during the hypersensitive reaction to Pseudomonas syringue pv. syvingae. Plant Physiol 98: 1304-1309

Tsuji J, Zook M, Somerville SC, Last RL, Hammerschmidt R (1993) Evidence that tryptophan is not a direct biosynthetic intermediate of camalexin in Arabidopsis thaliana. Physiol Mo1 Plant Pathol 43: 221-229

Widholm JM (1972a) Anthranilate synthetase from 5-methyltryp-

107: 77-85 tophan-susceptible and -resistant cultured Daucus carota cells. Biochim Biophys Acta 279 48-57

Widholm JM (1972b) Cultured Nicotiana tabacum cells with an altered anthranilate synthase which is less sensitive to feedback inhibition. Biochim Biophys Acta 261: 52-58

Yanofsky C, Crawford IP (1987) The tryptophan operon. 1n FC Neidhardt, JL Ingraham, KB Low, B Magasanik, M Schaechter, HE Umbarger, eds, Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol 2. American Society for Microbiology, Washington, DC, pp 1453-1472

Zhao J, Last RL (1995) Immunological characterization and chlo- roplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thalinna. J Biol Chem 270 6081-6087