Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid

Abstract

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

Figure 1.

TLC Analysis of Amino Acid Conjugates of IAA Synthesized by Recombinant GST:GH3s. (A) Assays for enzymatic formation of IAA-Asp with indicated reagents included in the reaction mixtures. The JAR1 enzyme conjugates amino acids to JA. Standards are shown in the two left lanes. (B) Evaluation of GH3.6 enzyme reactions with 20 amino acids (single letter abbreviations). (C) Assays for GH3.2, GH3.3, GH3.4, GH3.5, and GH3.17 reactions with the indicated amino acids. The spot near the origin at the bottom for the reactions with Trp is the unreacted amino acid. All plates were stained for indoles with van Urk-Salkowski reagent as described in the text.

Figure 2.

Adenylation Activity of IAA-Amido Synthetases on Several Auxins. Percentage of total radioactivity exchanged into ATP for each substrate is expressed as the mean of three independent experiments. Bars indicate standard errors. Cl-IAA, 4-chloro-3-indole acetic acid.

Figure 3.

Quantitation of IAA and IAA-Asp in the Wild Type and dfl1-D. Hormone level in 2-week-old Arabidopsis seedlings and 4-week-old leaves is shown as pg/g tissue fresh weight (FW) obtained from extraction of three independent tissue samples. Error bars indicate se of the means, and asterisks denote significant difference from the wild type (P < 0.05).

Figure 4.

Root Inhibition by Several Auxins in dfl1-D and Wild-Type Arabidopsis. Seedling roots were grown in agar media for 9 d with the indicated concentration of inhibitor. Inhibition is expressed as a percentage of each genotype grown in the absence of inhibitor. Error bars indicate 95% confidence intervals for each ratio of the means (n = 18 to 27 seedlings/data point).

Figure 5.

Analysis of DNA Insertion Mutants for IAA-Amido Synthetase Genes. (A) Insertion sites (open triangles) for T-DNAs (gh3.5-1 and gh3.17-1) or transposons (gh3.1-1 and gh3.2-1) were verified by amplification of genomic DNA using primers described in the text. Arrows indicate primer orientation. T-DNA mutants contain inverted tandem insertions yielding Lba1 primer sites at each end. Closed bars, coding regions; open bars, introns. (B) Auxin inhibition of root growth in insertion mutants. Inhibition and errors are expressed as in Figure 4 (n = 23 seedlings/data point). T-DNA mutants and transposon mutants are in the Col and Ler backgrounds, respectively.

Figure 6.

Phylogenetic Analysis of Plant GH3 Proteins. Proteins are identified by a two-letter genus/species designations followed by the gene name or database accession number for the nucleotide sequence from which the protein was translated. The Arabidopsis GH3 nomenclature is as described in the text. Le U144810 was constructed from unigenes SGN-U144810 build 2 (5′ end) and SGN-U54065 build 3 (3′ end) with ∼700 nucleotides of identical overlapping sequence (www.sgn.cornell.edu). The boxed enzyme JAR1 conjugates amino acids to JA. Dotted lines separate enzymes categorized as Group I, II, or III, based on sequence similarity and enzymatic activity (Staswick et al., 2002; Staswick and Tiryaki, 2004). Bootstrap values (1000 replicates) for individual nodes are represented by dots (≥95%), open circles (50 to 94%), or unlabeled (<50%). At, Arabidopsis thaliana; Cc, Capsicum chinense; Gm, Glycine max; Le, Lycopersicon esculentum; Nt, Nicotiana tabacum; Os, Oryza sativa; Pp, Physcomitrella patens.

Figure 7.

The Role of Amido Synthetase and Hydrolase Activity in IAA Homeostasis. Dotted arrow indicates transcriptional activation for several IAA-amido synthetase genes. The specific role of other IAA-amino acid conjugates in plants is unclear.