Arabidopsis thaliana GH3.15 acyl acid amido synthetase has a highly specific substrate preference for the auxin precursor indole-3-butyric acid

Abstract

Various phytohormones control plant growth and development and mediate biotic and abiotic stress responses. Gretchen Hagen 3 (GH3) acyl acid amido synthetases are plant enzymes that typically conjugate amino acids to indole-3-acetic acid (IAA) or jasmonic acid (JA) to inactivate or activate these phytohormones, respectively; however, the physiological and biological roles of many of these enzymes remain unclear. Using a biochemical approach, we found that the GH3.15 (AtGH3.15) preferentially uses indole-3-butyric acid (IBA) and glutamine as substrates. The X-ray crystal structure of the AtGH3.15·AMP complex, modeling of IBA in the active site, and biochemical analysis of site-directed mutants provide insight on active site features that lead to AtGH3.15's preference for IBA. Assay-based analysis of AtGH3.15-overexpressing lines indicated that their root elongation and lateral root density were resistant to IBA treatment but not to treatment with either IAA or JA. These findings suggest that AtGH3.15 may play a role in auxin homeostasis by modulating the levels of IBA for peroxisomal conversion to IAA. Analysis of promoter-driven yellow fluorescent protein reporter lines revealed that is expressed at significant levels in seedlings, roots, and parts of the siliques. We conclude that AtGH3.15 is unique in the GH3 protein family for its role in modifying IBA in auxin homeostasis and that it is the first GH3 protein shown to primarily modify a plant growth regulator other than IAA and JA.

Modification of auxinic phenoxyalkanoic acid herbicides by the acyl acid amido synthetase GH3.15 from Arabidopsis

“…S2). The amino acid profile was the same for AtGH3.15 with 2,4-DB as with IBA (33) with cysteine, histidine, methionine, glutamine, and tyrosine having the highest activity. Steady-state kinetics with cysteine, histidine, methionine, glutamine, and tyrosine were determined and confirm that, as with IBA, glutamine is the preferred amino acid for AtGH3.15 with 2,4-DB (Table 2).…”

“…The auxinic herbicides 2,4-D and 2,4-DB mimic the biological activity of the endogenous auxins IAA and IBA, respectively. Previous biochemical studies identified IAA-and IBA-specific GH3 acyl acid amido synthetases from Arabidopsis (26,32,33). Using purified recombinant protein, we examined the ability of the IAA-modifying AtGH3.1, AtGH3.2, AtGH3.5, and AtGH3.17 (32)…”

“…Kinetic parameters for IAA with AtGH3.1, AtGH3.2, AtGH3.5, and AtGH3.17 were published previously (32). Kinetic parameters for IAA and IBA with AtGH3.15 and for IBA with the other proteins were published previously (33). Average values Ϯ S.D.…”

“…In plants, the Gretchen Hagen 3 (GH3) acyl acid amido synthetases conjugate amino acids to carboxylic acid-containing hormones, such as jasmonic acid, IAA, and the endogenous auxin indole-3-butyric acid (IBA), to regulate plant growth, seed development, light signaling, and pathogen responses (25)(26)(27)(28)(29)(30)(31)(32)(33). GH3 proteins catalyze the adenylation of the carboxylate on these molecules to form an acyl-AMP intermediate, which undergoes nucleophilic attack by an amino acid to yield the conjugated product (28,30).…”

Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity

“…The levels of GH3.14, GH3.15 and GH3.17 increased upon flg22 exposure, the first significantly 1.2 fold. GH3.15 (AT5G13370) function in conjugating the IAA precursor IBA has just recently been elucidated (Sherp et al, 2018). GH3.14 (AT5G13360), the neighboring gene, does not shown any significant sequence homology indicating a potentially different uncharacterized function in PTI.…”