Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

Abstract

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.

Figure 1.

Purine biosynthesis pathway, structures of compounds used in this study, and effect of DAS734 on plants. A, The purine biosynthesis pathway. Abbreviations not defined in the text are as follows: PRA, phosphoribosylamine; AdS, adenylosuccinate; APRT, adenine phosphoribosyltransferase; ADA, AMP deaminase. The X symbols denote the sites of inhibition of DAS734 (GPRAT) and hadacidin (AdSS). B, Chemical structures of compounds used in this study. C, Effect of 1 kg/ha DAS734 on morningglory (top) and Arabidopsis (bottom) 10 d after greenhouse application. Note the bleaching of newly emerged leaves. D, Effect of DAS734 on Arabidopsis seedlings in plate tests.

Figure 2.

Effects of DAS734 on wild-type (Col-0) and DAS734-resistant Arabidopsis lines. A, Reversal by adenine of root growth inhibition by DAS734. Arabidopsis seedlings were grown in the presence of DAS734 and root lengths measured after 9 d. Typical root length (±sd) of untreated control seedlings was 17 (±1.2) mm. Adenine (185 μm) was added to the growth media in the samples labeled “+.” B, Dose response of three DAS734-resistant mutant lines to DAS734. The dashed line represents 50% inhibition of root growth. Data points are the average (±sd) of five individual root measurements. C, Effect of increasing concentrations of DAS734 on wild-type and resistant line R04, R08, and R06 seedlings grown in agarose media. Each plate contains a series of 3-fold dilutions of DAS734 starting at 72 μm, as shown in the top left plate containing wild-type (Col-0) seedlings. An untreated control well for each line is shown to the right of each plate. Based on root growth measurements, the resistance levels to DAS734 for R04, R08, and R06 are 6-fold, 56-fold, and 145-fold, respectively. The difference in resistance of the mutant lines to the phytotoxic effects of DAS734 on the cotyledon and shoots is also apparent. D, Effect of DAS734 applied as a 1 kg/ha postemergent spray to greenhouse-grown Arabidopsis plants. Plants were sprayed at the five- to six-leaf rosette stage and photographed 10 d later.

Figure 3.

Annotated polypeptide sequence of AtGPRAT2 showing sites of mutations conferring resistance to DAS734. For heterologous expression in E. coli, the AtGPRAT2 polypeptide was truncated by 75 residues, resulting in Asn-76 being converted to a starting Met residue. The conserved Cys that becomes the N-terminal active site residue after autoprocessing is marked. Mutations that were found by sequencing of the AtGPRAT2 genes from the DAS734-resistant mutant lines are shown above the sequence. The resistance level in seedling assays for the lines carrying the mutation is indicated in parentheses. Residues that are identical with the B. subtilis and E. coli GPRAT sequences are shaded gray. Residues within 10 Å of the active site thiol residue in the B. subtilis GPRAT crystal structure, PDB ID 1AO0 (Chen et al., 1997), and within 10 Å of the 6-diazo-5-oxonor-Leu-derivatized active site Cys in the E. coli GPRAT structure, PDB ID 1ECC1 (Krahn et al., 1997), are denoted with red dots below the sequence. These residues are therefore at or close to the glutaminase site of GPRAT. Residues within 10 Å of the bridging oxygen of the Rib pyrophosphate in GMP in the B. subtilis 1AO0 structure and in the carbocyclic PRPP analog in the E. coli 1ECC1 structure are denoted with blue dots below the sequence. These residues are therefore at or close to the PRPP catalytic site. The four Cys residues that are equivalent to the ligands for the 4Fe-S cofactor in B. subtilis GPRAT are marked.

Figure 4.

Expression of AtGPRAT2 in E. coli, effect of DAS734 on recombinant wild-type and mutant R264K AtGPRAT2 enzyme activity, and effect of purine nucleotides on wild-type and mutant enzymes. A, SDS-PAGE analysis of extracts from E. coli cells expressing AtGPRAT2 induced by the addition of 75 μm isopropyl-d-thiogalactopyranoside. A control extract with the empty pET26b vector and extracts from cells with expression plasmids containing from two separate AtGPRAT2 clones are shown The arrow denotes the presence of a new band at approximately 54 kD in the AtGPRAT2-expressing extracts. B, Inhibition of recombinant wild-type AtGPRAT2 and mutant enzyme R264K by DAS734. Extracts of E. coli expressing AtGPRAT2 were assayed by monitoring the PRPP-dependent production of Glu in the presence of increasing concentrations of DAS734. The level of activity is expressed relative to an untreated control reaction. Enzyme extracts were preincubated with DAS734 for 10 min prior to addition of substrates. The I₅₀ for DAS734 inhibition in this experiment was 0.5 μm. Subsequent experiments with a number of enzyme preparations gave I₅₀ values of approximately 0.2 μm. C, Time course of inhibition of wild-type and mutant enzymes. Substrates and 20 μm DAS734 were simultaneously added to AtGPRAT2 solutions and aliquots of the reaction mixture were analyzed for PRPP-dependent Gln production over time. Shown are wild-type AtGPRAT2 with no DAS734 (white circles), wild type plus 20 μm DAS734 (black circles), AtGPRAT2 R264K with no DAS734 (white boxes), and R264K plus 20 μm DAS734 (black boxes). D, Wild-type AtGPRAT2 activity was assayed in the presence of various nucleotides, alone or in pairs. All nucleotides were assayed at a concentration of 5 mm; thus, the total nucleotide concentration in the paired combinations was 10 mm. E, Dose response of wild-type AtGPRAT2 (solid lines) and AtGPRAT2 R264K (dashed lines) to 1:1 mixtures of AMP/ADP and AMP/ATP. Wild-type AtGPRAT2 with AMP/ADP (white circles), wild-type AtGPRAT2 with AMP/ATP (white boxes), AtGPRAT2 R264K with AMP/ADP (black circles), and AtGPRAT2 R264K with AMP/ATP (black boxes) are shown.

Figure 5.

Position of mutations conferring resistance to DAS734 mapped onto the structure of B. subtilis GPRAT (PDB ID 1AO0) from Chen et al. (1997). The residues in B. subtilis GPRAT that are equivalent to the mutation sites conferring resistance to DAS734 in AtGPRAT2 in the alignment in Supplemental Figure S3 are highlighted in red. The location of the Gln site can be identified by the N-terminal active site Cys residue (red dot). A GMP molecule (in blue) occupies the PRPP catalytic site and an ADP molecule occupies an adjacent allosteric site.

Figure 6.

Reversal of bleaching of atd2 mutant seedlings by adenine. Eight-day-old atd2 seedlings on media with no supplementation (A), 184 μm hypoxanthine (B), or 185 μm adenine (C) are shown. The atd2 bleached mutant phenotype is reversed to a normal green color by addition of adenine, whereas addition of hypoxanthine has no effect.