title: 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.
authors.affiliation: Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68583, USA. pstaswick1@unl.edu
authors.forename: Paul E
authors.initials: PE
authors.lastname: Staswick
authors.forename: Bogdan
authors.initials: B
authors.lastname: Serban
authors.forename: Martha
authors.initials: M
authors.lastname: Rowe
authors.forename: Iskender
authors.initials: I
authors.lastname: Tiryaki
authors.forename: Marién T
authors.initials: MT
authors.lastname: Maldonado
authors.forename: Mitsa C
authors.initials: MC
authors.lastname: Maldonado
authors.forename: Walter
authors.initials: W
authors.lastname: Suza
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D006706:Homeostasis; D007210:Indoleacetic Acids / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D018517:Plant Roots / Q000254:growth & development / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D007210:Indoleacetic Acids; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C421912:indole-3-acetic acid synthase; C030737:indoleacetic acid