Copyright 0 1997 by the Genetics Society of America

An Allelic Series of Blue Fluorescent trpl Mutants of ArabidqPsis thalianu

Alan B. Rose,' Jiayang L i 2 and Robert L. Last

Boyce Thompson Institute for Plant Research and Section of Genetics and Development, Cornell University, Ithaca, New York 14853-1801

Manuscript received June 14, 1996 Accepted for publication September 27, 1996

ABSTRACT Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic

selections and fluorescence screens. Each was shown to contain a recessive allele of trpl, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trpl mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trpl alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trpl mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.

M UTANTS blocked in a single step of a biochemi- cal pathway are valuable tools in the elucidation

of metabolic pathways and their regulation in microor- ganisms. The study of metabolism in plants has been hampered by difficulties in obtaining auxotrophic mu- tants, the most common general class of mutant in microbes. Possible explanations for the rarity of plant auxotrophs include duplication of structural genes, biochemical redundancy that can provide more than one way to make a substance, and the requirement of a needed metabolite during gamete formation or embryogenesis (SCHNEIDER et al. 1989). In a limited number of cases these obstacles were overcome by screening large numbers of mutagenized seeds under selective conditions that strongly favor the growth of mutants over wild-type plants (BRAAKSMA and FEENS- TRA 1982; DOLFERUS and JACOBS 1984; HAUGHN and SOMERVILLE 1986; MOFFATT and SOMERVILLE 1988).

The tryptophan biosynthetic pathway of Arabidopsis thaliana has been unusually amenable to genetic analy- sis. This pathway has relevance beyond its role in pro- tein synthesis because it also serves as the source of numerous important secondary metabolites such as the growth hormone auxin (indole acetic acid), antimicro- bial phytoalexins, and indole glucosinolates. Mutations in four of the seven enzymatic activities that constitute

Corresponding author: Alan B. Rose, Section of Molecular and Cellular Biology, University of California, Davis, CA 956168535. E-mail: abrose@ucdavis.edu

h e n t addmss: Section of Molecular and Cellular Biology, Univer- sity of California, Davis, CA 956168535.

Present address: Institute of Genetics, Academia Sinica, Beijing 100101. China.

the tryptophan biosynthetic pathway were isolated from selections for Arabidopsis seedlings resistant to methyl- or fluoro-analogues of the tryptophan precursors an- thranilate or indole (LAST and FINK 1988; LAST et al. 1991; BARCZAK et al. 1995; LI and LAST 1996). These compounds are thought to be harmless unless con- verted into toxic tryptophan analogues by the enzymes of the tryptophan pathway. Mutants unable to effi- ciently catalyze this conversion due to a defect in a tryptophan pathway enzyme can survive normally lethal concentrations of substituted pathway intermediates.

The first Arabidopsis tryptophan pathway mutation was isolated from a selection for resistance to 5-methyl- anthranilate (5MA, LAST and FINK 1988). Homozygous t rp l - I plants are auxotrophic for tryptophan due to a defect in the enzyme phosphoribosylanthranilate trans- ferase (PAT), which converts anthranilate and phos- phoribosylpyrophosphate into phosphoribosylanthrani- late and inorganic pyrophosphate. They grow very slowly in soil, have a bushy and stunted morphology, and are virtually sterile due to floral abnormalities. In addition, the t q l - 1 mutants accumulate anthranilate p- glucosides, which renders the entire plant fluorescent under UV light (LAST and FINK 1988), a phenotype that was used to isolate the t rp l - IO0 mutation (ROSE et al. 1992). Unlike t q l - 1 , the fluorescent t rp l -100 plants are prototrophic for tryptophan and have normal growth rates, appearance, and fertility.

The dramatically different morphologies of the t rp l - 1 and t q l - I 0 0 plants led us to seek additional t rp l mu- tants. An allelic series would allow us to determine if the very different properties of the t rp l -1 and t r p l - I 0 0 mutants represent two points on a broad spectrum of

Genetics 145: 197-205 (January, 1997)

198 A. B. Rose, J. Li and R. L. Last

characteristics, or if the phenotypes of either are un- usual or allele-specific. Furthermore, because PATl (the gene defective in the trpl mutants) is apparently the only single-copy gene of tryptophan biosynthesis (ROSE et al. 1992), mutations in this step are particularly useful for exploring the consequences of reduced flux through this key metabolic pathway. A third reason for analyzing trpl mutations was to begin to identify resi- dues important for protein function or stability; no analysis of mutations that affect PAT from any organism is published.

Here we describe an allelic series of trpl mutations isolated from screens for fluorescent plants or selec- tions for resistance to anthranilate analogues. The properties of the trpl mutants suggest that as PAT en- zyme activity is decreased by successively stronger muta- tions, the first phenotype to appear is methylanthrani- late resistance, then bright fluorescence, followed by auxotrophy (with abnormal growth and fertility), and finally reduced viability.

MATERIALS AND METHODS

Mutant isolation and plant growth conditions: The Colum- bia (Col-0) and Landsberg erecta (Ler) ecotypes of Arabidopsis were mutagenized with EMS or nitrosomethylurea (NMU) as described (BARCZAK et al. 1995). The resulting M2 seeds were subjected to selections for resistance to 5MA or &methylan- thranilate ( 6 M , LAST and FINK 1988; LI and LAST 1996) or were screened for fluorescence under short- or long-wave- length UV light (ROSE et al. 1992). The self-crossed progeny of these mutants were outcrossed to wild type at least once, and seeds were submitted to the Arabidopsis Biological Re- source Center at Ohio State (http://aims.cps.msu.edu/aims/) with the following accession numbers: trpl-1, CS8157; trpl-2,

101, CS8162; trpl-102, CS8163; trpl-103, CS8164; trpl-104, CS8165; trpl-105, CS8166; trpl-106, CS8167. A mutant was classified as an auxotroph or a prototroph based on it's ability to grow on agar medium lacking tryptophan under high-light conditions as described (LAST and FINK 1988). Auxotrophic plants were transplanted to soil-less mix (Cornell mix, LANDRY et al. 1995) after germination and growth for 2-3 weeks on PNS supplemented with 50 p~ tryptophan. In most genetic crosses the pollen recipient carried the recessive gll mutation (KOOFWNEEF et al. 1982) allowing confirmation of outcrossing by the presence of trichomes on the F1 plants. All other condi- tions for the growth and genetic manipulation of plants were as previously described (BARCZAK et al. 1995).

Analysis of fluorescence: Rosette leaf extracts from plants grown in soil were prepared and analyzed for fluorescent compounds by modification of published methods (LAST and FINK 1988). The auxotrophic mutants were allowed to grow for 2 months before harvest, and cauline leaves were also used if plants lacked sufficient rosette leaves. Fluorescence photographs were taken under short-wave UV light using Ko- dak Ektar 1000 film and a Wratten gelatin filter No. 47 (East- man Kodak, Rochester, N u ) .

Enzyme assays: The PAT and phosphoribosylanthranilate isomerase (PAI) enzyme activities of mutant and wild-type plants were assayed as described (LAST and FINK 1988), with the following modifications. The reactions contained 10 p M anthranilate and 25 pL of bacterial extract. For PAT assays

CS8158; tql-3, CS8159; tql-5, (36247; tql-100, CS8161; t q l -

TABLE 1

crpl alleles

Allele Method of Tryptophan Fluorescence designation" isolationb requirement units'

trpl-I trpl-2 tql-3 tql-5 trpl -I 00 trpl-101 trpl -I 02 tql-103 tql-104 tql-I05 te1-106

5MAd U V d

wd wd U V d

wd w d

w" 5MAd 6MA" 6MA"

+ 5000 + 4000 + 4700 + 5600

1000 1100 850

- 1200 200 85

900

- -

-

- - -

a trpl-3 is in the Ler ecotype. All other mutants are Col-0. '5MA and 6MA, resistance to 5- or &methylanthranilate,

'One unit is the fluorescence equivalent to that from 1 respectively; U V , screen for UV fluorescence.

nmol anthranilic acid per g fresh weight. From EMS mutagenesis.

"From NMU mutagenesis.

of wild-type plants and all PAI assays, 50 pL of plant extract was used per reaction, which were stopped after 15 or 30 min. PAT activity in t q l mutants was assayed with 200 pL of plant extract and the reactions times were 30 or 60 min.

Molecular analyses: RNA was extracted, blotted to nitrocel- lulose, and probed with a PATl cDNA (pAR129) as described (ROSE and LAST 1997). Band intensities were quantified by PhosphoImager (Molecular Dynamics model #400A, Sun- nyvale, C A ) and normalized to the amount of ethidium bro- mide-stained RNA in each lane. Immunological methods were as previously published (ZHAO and LAST 1995). Total genomic DNA from the trpl-I , trpl-3, trpl-100, and trpl-102 mutants, and Col-0 and Lerwild-type plants was digested to completion with BamHI. DNA in the 4-8 kb size range was ligated into X Z A P Express (Stratagene, LaJolla, CA). The PATl gene from each library was identified using the digoxigenin-labeled PATl cDNA insert from pNN423 (ROSE et al. 1992). Single- stranded DNA was sequenced using MultiWell rapid sequenc- ing kits (Amersham, Arlington Heights, IL).

RESULTS

Isolation of trpl mutants: The very different growth rate, morphology, and fertility of the t q l - 1 and t q l - 100 mutant plants (LAST and FINK 1988; ROSE et al. 1992) suggested that the consequences of trpl muta- tions are potentially quite diverse. More alleles of tqb1 were sought to explore the range of phenotypes possi- ble. A total of nine new mutations were isolated that were subsequently identified as alleles of t q l , and these are listed in Table 1. Three of the mutations were iso- lated from selections for resistance to 5MA ( tq l -104) or 6MA (trpl-105 and trpl-106), and the remaining six were identified in fluorescence screens. The conclusion that each mutation is a trpl allele is based on genetic and biochemical analysis, as described below. Table 1 includes all of the fluorescent isolates obtained and

Arabidopsis Tryptophan Mutants 199

TABLE 2

Results of crosses with wild type

Cross” Type Total Nonfluorescent Fluorescent % Fluorescent

TRPl/TRPl X trpl-2/tql-2 F1 2 2 0 0 Fz 98 1 783 198 20*

F2 478 419 59 12*

F2 982 938 44 4* tql-lOO/tql-100 X TRPl/TRPl F1 10 10 0 0

F2 514 400 114 22

F2 100 87 13 13*

FP 100 78 22 22

F2 172 141 31 18*

F2 180 133 47 26

F2 390 289 101 26

F2 396 342 54 14*

TRPl/TRPl X tq1-3/tq1-3 F1 8 8 0 0

TRPl/TRPl X tq l -5 / tq l -5 F1 3 3 0 0

TRPl/TR.Pl X t7pl-l0l/trpl-101 FI 11 11 0 0

TRPl/TRPl X t ~ 1 - 1 0 2 / t ~ 1 - 1 0 2 F1 16 16 0 0

TRPl/TRPl X trpl-lO3/tqI-l03 F1 9 9 0 0

TRPl/TRPl X tql-104/tq1-104 F1 40 40 0 0

tql-l05/tql-105 X TRPl/TRPl F1 8 8 0 0

t~1-106/trp1-106 X TRPI/TRPI F1 8 8 0 0

* Chi-square P > 0.95, based on an expected frequency of 25%. “Fluorescent mutants were crossed with wild type to generate F1 seeds (pollen recipient is listed first). The heterozygous F1

plants were allowed to self-pollinate to yield F2 progeny-

only those methylanthranilate resistance-conferring mu- tations that are allelic to trpl.

The trpl mutants can be classified into two very differ- ent groups on the basis of several phenotypic character- istics. As is the case for the canonical trpl-1 mutant, the auxotrophic mutants trpl-2, tql-3, and tql-5 grow very slowly in soil and have reduced apical dominance and fertility. The trpl-5 mutants are the most severely af- fected of the auxotrophs isolated, as judged by the very slow growth and complete sterility of homozygous plants. In contrast, the trpl-101, tql-102, trpl-103, t rpl- 104, trpl-105, and trpl-I06mutants, like trpl-100, grow normally without tryptophan and are morphologically indistinguishable from wild type.

Genetic characterization of trpl mutants: To confirm that the observed phenotypes are caused by single reces- sive mutations, each of the mutants was crossed to wild type, and individuals from the F1 and F2 generations were scored for fluorescence or resistance to 6MA. The observation that the F1 plants in each of the crosses showed the wild-type phenotype demonstrates that all of the mutations are recessive (Table 2). In crosses with the prototrophic trpl mutants, approximately onequar- ter of the F2 progeny derived from self-pollination of the F1 plants were fluorescent or 6MA-resistant, consis- tent with the idea that the phenotypes are caused by mutations in a single gene.

The crosses involving the auxotrophic mutants show segregation that is most easily explained as being monogenic, although the ratio of wild-type:mutant

plants in the F2 generation is somewhat higher than the 3:l expected for a single recessive mutation. The frequency of fluorescent F2 individuals was 20% and 12% for crosses with the trpl-2 and trpl-3 mutants, respectively (Table 2). These values are well above the 6.25% predicted for a trait caused by two unlinked recessive mutations. A cross of the severely affected trpl-5 mutant to wild type gave a 22:l nonfluores- cent:fluorescent F2 ratio, possibly implicating a second mutation. Alternatively, this could be another example of segregation distortion associated with tryptophan auxotrophy, which in the previous case was ascribed to reduced viability of the mutant female gametophytes (NIYOGI et al. 1993).

Mutations other than trpl can be isolated from meth- ylanthranilate selections and fluorescence screens. For example, resistance to anthranilate analogues can arise from a defect in any step of the tryptophan pathway (R~DWANSKI and LAST 1995; LI and LAST 1996) or possi- bly from a failure to take up the selective agent. Plants with impaired PAI activity also accumulate anthranilate compounds (BENDER and FINK 1995; LI et al. 1995), and fluorescence could be caused by the accumulation of a substance unrelated to anthranilate, such as sinapic acid (CWPLE et al. 1992). Complementation crosses were performed with trpl-100, to determine which of the methylanthranilate-resistant or fluorescent proto- trophs contain trpl mutations. As shown in Table 3, F1 crosses show that the trpl-101, trpl-102, trpl-103, trpl- 104, trpl-105, and t~pI-106mutations all fail to comple-

200 A. B. Rose, J. Li and R. L. Last

TABLE 3

Results of crosses with VI-100

Cross" Type Total Nonfluorescent Fluorescent % Fluorescent

tq1-100/t7p1-100 x +/tq1-2 FI 126 73 53 42

tq1-100/tq1-100 x t q I - I O I / t q l - 1 0 1 FI 26 0 26 100 F.l 199 0 199 100

tql-loo/tql-loo x tq1-102/tq1-102 FI 17 0 17 100 F'2 200 0 200 100

F'L 213 0 213 100 1q1-100/tq11-100 X rqlI-104/trpl-104 Fl 40 0 40 100

F'L 135 0 135 100

F2 176 0 176 100 tq1-100/tq1-100 x tql-l06/tql-l06 FI 8 0 8 100

F2 199 0 199 100

t ~ l - 1 0 0 / ~ ~ 1 - 1 0 0 X +/ tq l -3 FI 153 85 68 44 t~ l - l00 / t~1 l - I00 X +/tq1-5 FI 130 61 69 53

L ~ l - l 0 0 / t ~ l - l 0 0 X t ~1 -103 / t~1 -103 FI 16 0 16 100

t ~ l - l 0 0 / t ~ l - l 0 0 X t ~ 1 - 1 0 5 / t ~ 1 - 1 0 5 Fl 8 0 8 100

" Pollen from the mutants was crossed with trjll-100plants to generate FI seeds (pollen recipient is listed first). The heterozygous FI plants from crosses with prototrophs were allowed to self-pollinate to yield F2 progeny.

ment tq1-100. As expected for allelic mutations, F2 anal- ysis indicated that these new alleles are very tightly linked to the trpl locus.

To test whether each of the auxotrophic mutations is allelic to trpl, pollen from plants heterozygous for the auxotrophic frp1 mutations was crossed onto trpl- 100 plants, and the number of fluorescent individuals in the F, generation was scored. Heterozygous donors were used because trpl auxotrophs make abnormal flowers with very little pollen, and repeated attempts to cross homozygous auxop-ophs to trpl-100 plants failed. As shown in Table 3, approximately half of the F1 prog- eny of these test crosses were fluorescent, confirming that each of the auxotrophs contains a trpl mutation that fails to complement the trpl-100 mutation. These results strongly suggest that the mutations listed in Ta- ble l are all alleles of t q 2 .

Accumulation of anthranilate compounds: Several trpl mutant characteristics can be compared to indirectly assess the degree to which the PAT enzyme is impaired and correlate these defects with growth of the plant. One such trait is the amount of anthranilate com- pounds they contain. An estimate of the relative accu- mulation was obtained by observing the fluorescence of cotyledons. As shown in Figure lA, all of the trpl mutants are more fluorescent than wild type, although the fluorescence of the trpl-104 mutants is weak and that of the trpl-105 mutants is barely detectable. To quantitate this phenotype, ethyl acetate extracts from leaves of each of the mutants were measured in a fluo- rescence spectrophotometer, with the results pre- sented in Table 1. The small amount of fluorescent material in a control extract made from wild-type plants (12 units) indicates that virtually all of the fluo- rescence in the mutant extracts is a consequence of

the trpl mutations. As previously shown for tq l - I (LAST and FINK 1988), thin layer chromatographs of extracts with and without P-glucosidase treatment identify most of the fluorescent material as anthrani- late, predominantly in the form of an anthranilate 0- glucoside (data not shown).

PAT enzyme assays: Enzyme assays were performed to provide direct biochemical support for the hypothe- sis that the fluorescent mutants have defects in PAT and to test the mutants for differences in residual PAT activity. As predicted from the complementation re- sults, all of the trpl mutants examined have significantly reduced PAT activity (Table 4). Only the least fluores- cent of the prototrophic mutants, trpl-104 and trpl-105, had detectable PAT activity (7% and 5% of wild type, respectively). The PAT activity of each of the other pro- totrophs was below the limit of detection, which we

FIGURE 1.-Fluorescence and 6MA-resistance of tql mu- tants. (A) Eight-day old seedlings under UV light. (B) Plants grown for 20 days on media containing 250 p~ 6MA and 25 ~ L M tryptophan.

Arabidopsis Tryptophan Mutants 201

TABLE 4

Specific activities of tryptophan pathway enzymes

Phosphoribosvlanthranilate Phosphoribosylanthranilate Strain transferase isomerase

Wild type 810 2 120 2800 ? 500 lrpl00 ND 2700 t q 1 - I O 1 ND 3600 t9l-102 ND 3800 tql-I03 ND 4100 tql -104 65 ? 5 3100 2 300 t q l - I 05 40 2 4 2800 t q l - IO6 ND 4400

Specific activities are reported in pmol of anthranilate per minute per mg protein ? standard deviation. ND, not detectable (< lo unit$).

estimate to be 1% of wild-type activity. Although a de- fect in the next reaction of the tryptophan pathway (PAI) should also cause an accumulation of fluorescent anthranilate compounds, these mutants all have wild- type levels of PAI activity (Table 4). These observations confirm that trp1 mutations affect the PAT enzyme.

Resistance to 6MA: A prediction based on the fact that the least fluorescent trp1 mutant (tql-105) and a brightly fluorescent auxotroph ( t r p l - I ) were both iso- lated from anthranilate analogue selections is that all trpl mutants should be resistant to methylanthranilate. This was confirmed by growing seedlings on media con- taining 250 /AM 6MA, a concentration that strongly in- hibits the growth of wild-type plants. All of the mutants are fully resistant to this concentration of 6MA, as shown in Figure 1B.

PATl protein and mRNA in trpl mutants: The lack of PAT activity in the trpl mutants could be due to a decreased amount of PAT enzyme or to PAT proteins with reduced catalytic activity. To differentiate between these possibilities, the amount of protein in each of the trpl mutants was determined by immunoblotting with polyclonal anti-PAT antibodies (ZHAO and LAST 1995). As shown in Figure 2A, tql-3 lacks detectable PAT pro- tein and the tql-102 mutant has reduced levels, while the other trpl mutants have PAT protein of the same

- TSA B

FIGURE 2.-PAT protein and PATI mRNA in the trpl mu- tants. (A) Immunoblot of total protein probed with antibodies that recognize PAT (top) or the a subunit of tryptophan synthase (TSA) as a loading control (bottom). (B) RNA gel blot probed with a PATI cDNA.

mobility and abundance as wild-type plants. This indi- cates that the primary defect in most of the tqll mutants is in the activity rather than the accumulation of PAT enzyme.

To ask whether the reduced protein levels in the t q l - 3 and tq11-102 mutants is caused by low mRNA accumu- lation, a blot of total RNA from each of the trpl mutants was probed with a PAT1 cDNA. As shown in Figure 2B, all of the trpl mutants contain as least as much PAT1 mRNA as wild type, and the auxotrophic tql mutants trpl-I, trpl-2, and trpl-5 have approximately threefold more. The normal PAT1 mRNA accumulation and re- duced protein abundance of the trpl-3 and tql-102 mutants suggests a defect in translation of PATl mRNA or stability of the mutant PAT proteins.

Sequence of mutant alleles: Four representative al- leles of t q l were chosen for molecular analysis to corre- late phenotypic consequences with lesions in specific residues of the PAT protein. These alleles were chosen because the trpl-1 and trpl-100 mutants are the canoni- cal members of their phenotypic class, and the t9I-3 and tql-102 mutants have reduced PAT protein. The trpl-I , tq1-100, and trpl-102mutations are single nucle- otide transitions in the PATI coding region that are predicted to cause an amino acid change, as shown in Figure 3. As expected for mutations caused by an alkylating agent (EMS), all are G to A or C to T transi- tions. Each alters a different codon, yet in all cases the affected amino acid is highly conserved: the residues affected by the trpl-I (Gly 161 to Arg), tql-100 (Ser 224 to Asn), and trpl-102 (Thr 277 to Ile) mutations are found at the analogous locations in 15, 13, and 16, respectively, of the 17 PAT enzymes for which data are available. Furthermore, none of the amino acids cre- ated by a trpl mutation are found in the analogous position in any of the 17 species.

In contrast, the tql-3 allele of PAT1 contains two single nucleotide differences from the wild-type Ler PAT1 sequence. One is in the first intron of PATI, while the other changes residue 115 from arginine to gluta-

A. B. Rose, J. Li and R. L. Last 202

A

B TRPl

t w l - 3

TRP 1

trnl-1

115

ttgamt ggtttaggt TRpl GATCGG GIT

ttgamt ggtttaggt t p l - 3 GATCAG GTT f D R V

4 Q

161 V G L

224 G S A

277 K T V

GTG GGG l T A TRPl GGAAGT GCT TRPl AAA ACT G I T 4 4 4

GTGAGGTTA t p l - 1 0 0 GGAAATGCT t r ~ 1 - 1 0 2 A A A A l T G l T R N I

FIGURE 3.-DNA sequence changes of the trpl-1, trpl-3, tql-100, and tql-102 mutations. (A) Schematic representa- tion of the location of the tql mutations within the PATl gene. H, exons; -, introns and flanking sequences. (B) De- tails of the trpl mutations. Intronic sequences are in lower- case, protein coding sequences are in uppercase, and the bases affected by mutation are in bold. Amino acids are in the single letter code, with the residue changed by mutation numbered.

mine. The affected amino acid lies near the predicted junction between the transit peptide and the mature portion of the PATl protein and is not evolutionarily conserved.

PATl is unusually divergent in two Arabidopsis eco- types: The wild-type Col-0 and Ler accessions of Arabi- dopsis are closely related, and their origins can be traced to the same segregating population ( R ~ D E I 1992). It was therefore surprising to find an unusually high degree of divergence in the PATl gene from these two ecotypes. In the 2627-bp Col-0 and 2534bp Ler PATl sequences compared in Figure 4, there are 90 nucleotide differences and 12 insertions/deletions ranging in size from 1 to 81 bp. Although the exons are less divergent than the introns (2.9% us. 7.7%), seven of the single base differences result in amino acid polymorphisms. Five of these residues and the three affected by a 9-bp insertion/deletion in the first exon are in the putative plastid transit peptide, a region that can presumably tolerate changes more readily than the rest of the enzyme. The two amino acid differences in the mature part of the protein (Col-0 Thr 124 is Ser and Val 234 is Ala in Ler) are chemically conservative changes in phylogenetically poorly conserved residues. Therefore, the mature PAT proteins from these two ecotypes are probably very similar in structure and func- tion, despite the large number of differences at the DNA level. It is intriguing that the nucleotide polymor- phisms in PAT1 are not uniformly distributed: there are more differences in the 5' half of the gene than in the 3' half. In fact, there are no differences at all within the 650 bp at the 3' end of the gene that include the

Arabidopsis Tryptophan Mutants 203

last three exons and two introns of PATl and 100 bp of 3' flanking sequences.

DISCUSSION

To appreciate fully the consequences of mutations at any locus, it is desirable to characterize an allelic series of mutants. We have done so for the t q l locus because of the unique characteristics of the defining alleles t q 1 - 1 and tql-100 (LAST and FINK 1988; ROSE et al. 1992). Nine new mutations were isolated and shown to be al- leles of t q l by complementation crosses, enzyme assay, and DNA sequencing. The allelic series of t q l muta- tions can be ranked in order based on residual PAT enzyme activity, fluorescence, tryptophan requirement, and viability. From the least severely affected to the most impaired, this ranking is as follows: tql-105 5 tql-104 < tql-100 M tql-101 M tyl-102 M tql-103 M

tql-106 Q tql-1 tql-2 tql-3 < tvl-5. Although mutations in the next step of the pathway (PAI) should yield blue fluorescent and 6MA-tolerant plants, PAI- deficient mutants were not identified, presumably be- cause this protein is encoded by three functional genes in Col-0 (BENDER and FINK 1995; LI et al. 1995).

The analysis of the new t q l alleles has increased our understanding of this step of the tryptophan pathway in several ways. Most importantly, we have identified an allele ( tq l -5) that is more severe than t q l - 1 and two (tql-104 and trpl-105) that are leakier than tql-100, thereby extending the range of phenotypes associated with tql mutations. The context provided by the new alleles reveals that the phenotypes of the trpl-1 and trpl- 100 mutants are typical for auxotrophs and prototrophs respectively, and are not allele-specific.

Second, the lack of t q l mutants with phenotypes intermediate between those of t q 1 - 1 and tql-100 (e.g., a moderately fertile auxotroph or a bushy, slow-growing prototroph), despite several methods of mutant isola- tion, suggests that the large difference in appearance between these groups is caused by relatively small differ- ences in PAT activity. The possibility that fluorescence and auxotrophy are due to separate mutations can be eliminated for the tvl-1 mutant because both pheno- types are corrected in transgenic plants by a fragment containing only the PATl gene (ROSE et al. 1992). If a certain threshold level of tryptophan (or a pathway- derived secondary metabolite) is required for growth, a mutant will either be a prototroph or an auxotroph depending on whether the amount of tryptophan it can manufacture is above or below this threshold. Appar- ently this critical level can be lowered by reducing the growth rate (and presumably the demand for trypto- phan): the tql-1, tq2-l and tq3-1 mutants are usually auxotrophs but do not require tryptophan when grown under low light conditions (LAST et al. 1991; E. R. RAD- WANSKI and R. L. LAST, unpublished results).

A third noteworthy feature revealed by the analysis of the new trpl alleles is that PAT enzyme activity appar- ently is present in at least 10-fold excess in wild-type plants. The tql-104 and tql-105 mutants are only weakly fluorescent with 5-7% of in vitro wild-type PAT enzyme activity, and the remaining prototrophs grow normally and are fully fertile with < 1 % of normal PAT activity. Metabolic flux theory predicts that almost all enzymes will appear to be in excess, and therefore rela- tively large decreases (or increases) in the level of indi- vidual pathway enzymes will have minor effects on flux through that pathway (KACSER and BURNS 1995). This was found to be the case for the tryptophan pathway in yeast, although tryptophan production was more sensi- tive to a reduction in the level of PAT than any other pathway enzyme (NIEDERBERGER et al. 1992). However, a decrease in enzyme activity of 299% usually impairs a pathway significantly, although other exceptions in- clude Escherichia coli 0-galactosidase mutants (DEAN et al. 1986) and the leaky phytochelatin synthasedeficient cadl-1 mutants of Arabidopsis ( HOWDEN et al. 1995).

A defect in the singlecopy PATl gene that causes a complete block in this pathway, the only known source of indole, should be lethal due to starvation for trypto- phan and lack of the essential growth hormone auxin. It may be impossible to rescue a trpl null by supplying tryptophan because auxin is derived from a pathway intermediate, not tryptophan, in maize and Arabidopsis (WRIGHT et al. 1991; NORMANLY et al. 1993). Consistent with the hypothesis that PATl is an essential gene, none of the sequenced trpl alleles contain nonsense muta- tions. The alteration in the tql-102 allele presumably destabilizes the protein, leading to decreased PAT accu- mulation (Figure 2A). In the three analyzed mutants that contain detectable PAT protein, DNA sequencing revealed changes in evolutionarily conserved amino acids, which presumably have important roles in enzyme structure or catalytic function. The PAT crystal structure is not published (EDWARDS et al. 1988), preventing a critical assessment of the function of these residues.

The identification of two mutations in trpl-3 compli- cates our understanding of this allele. While the Arg to Gln amino acid replacement may not impair catalytic activity (because it affects a poorly conserved residue that lies very near the predicted amino terminus of the mature enzyme), it could destabilize the PAT protein or interfere with import into the plastid or transit peptide cleavage. Any explanation of how the intron mutation could prevent protein accumulation must account for the normal size and amount of PATl mRNA in the tql-3 mutants. We propose that the AG created by the mutation does not function as a 3' splice site because the resulting 17 nucleotide intron would probably be too small to be spliced (GOODALL and FILIPOWCZ 1990), and the predicted 93 nucleotide increase in PATl mRNA size is not seen (Figure 2B).

204 A. B. Rose, J. Li and R. L. Last

The 4.1% divergence of the PATl gene between the Col-0 and Ler ecotypes is the highest of all the Arabi- dopsis genes for which such a comparison is available. It has been estimated that genes from Col-0 and Ler differ by 0.4% on average (KONIECZNY and AUSUBEL 1993), and most published examples show a divergence of <0.2% (for examples see HANFSTINGL et al. 1994; MITA et al. 1995; REISER et al. 1995; SHIRLEY et al. 1995). In the tryptophan pathway, the TSAl gene encoding tryptophan synthase alpha subunit has only four single base polymorphisms in 3072 bp, and three of these differences are in 5' flanking sequences (RADWANSKI et al. 1996). The gene with the second highest known divergence between Ler and Col-0 is GA5 (GenBank accession numbers U20872 and U20873, respectively), which has 30 nucleotide changes and two insertions/ deletions within 1432 bp (2.2% divergence). Polymor- phisms are clearly distributed unevenly throughout the genome, and this nonuniformity is also apparent within individual genes. For example, most of the divergence is found in the 5' half of PATI, and 12 of the 15 ecotype differences in the 2.9-kb ADH gene are clustered in a 299-bp region (WFSTINGL et al. 1994).

The t q l mutants described here could be used in various applications, especially in conjunction with the cloned PATl gene. An auxotrophic t q l mutant would enable the use of the PATl gene as a selectable marker during plant transformation. Their ease of growth and bright fluorescence make t q l prototrophs suitable for many types of genetic schemes. Indeed, a visual screen for suppressors of the fluorescence of t q l - 1 0 0 plants was used to isolate the t q 4 mutants, which are leaky anthranilate synthase beta subunit alleles (NIYOGI et al. 1993). Because the weakest mutants are most similar to wild type, they might be the best genetic background for selections against the presence of the wild-type PATl gene using 6MA. Similar counter-selectable markers are central to the targeted disruption of genes in mice be- cause they provide a way to enrich for transgene integra- tions that result from homologous recombination (CA- PECCHI 1989).

We thank AMY CASSELMAN for her help with the visual screen for mutants, JIANMIN ZHAO for assistance with immunoblots, and Drs. ANNE BRITI, JIANMIN ZHAO, and LESILEE ROSE for comments on the manuscript. This work was supported by the Biotechnology Research and Development Corporation, the National Institutes of Health grant GM-43134, and a National Science Foundation Presidential Young Investigator Award No. DMB-9058134 to R.L.L.

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