IDENTIFICATION AND CHARACTERIZATION OF IAA OXIDASES AND THEIR ROLE IN IAA HOMEOSTATIC REGULATION IN ARABIDOPSIS
Peer, Wendy Ann
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Auxin is a crucial plant hormone that shapes and directs plant growth. Indole-3-acetic acid (IAA) is the predominant auxin in nature. Auxin regulates cell expansion and cell division in a dose dependent way. Therefore, plants evolved an extremely complex yet highly coordinated network to maintain auxin homeostasis, including IAA biosynthesis, transport, conjugation and oxidation. Among these, the least known process is IAA oxidation. Discovering how IAA is terminated is very important in completing the whole picture of IAA homeostatic regulation. By partial purification of IAA oxidases from Arabidopsis, we detected IAA oxidation activity from both microsomal fractions and soluble fractions. We first investigated the protein in microsomal fraction and identified one oxidase named as ACC oxidase 2 (ACO2), an ethylene synthetase that belongs to 2-oxoglutarate and iron (II) [2OG(Fe)] dependent dioxygenase family. In vitro enzyme assays with IAA showed that ACO2 could catabolize IAA and that the product had the same retention time as indole-3-carbinal (ICA), an decarboxylative IAA oxidation product. The same enzyme assay with the ACO2 homologues ACO3 was conducted, and ACO3 showed similar activity. An ACO2 loss-of-function allele showed ethylene related phenotypes, including longer hypocotyls and reduced apical hook angle in etiolated seedlings, and delayed bolting. Further, null aco2 mutants also showed reduced phototropic bending, a typical auxin related phenotype. These results indicate that ACO2 might be involved in both ethylene and auxin signaling. We also investigated the soluble IAA oxidases, AtDAO1 (DAO1) and AtDAO2 (DAO2). In vitro enzyme assays showed that both recombinant DAO1 and DAO2 have IAA oxidation activity and the product is the non-decarboxylated 2-oxindole-3-acetic acid (oxIAA), the major IAA metabolite observed under normal growth conditions. Analysis of the loss-of-function null allele dao1-1 showed that DAO1 is the predominant IAA oxidase and is responsible for 95% of oxIAA production in Arabidopsis seedlings. Dysregulation of IAA oxidation altered the IAA metabolism profile and causes accumulation of other IAA conjugates and a series of morphological alteration, including elongation of organs, increased lateral roots and delayed sepal opening. Investigation of expression patterns shows that DAO1 is a cytosolic protein that widely expressed throughout the plant, especially in the root tip, the pericycle of root, the cotyledon, and the sepal, highly correlating to the phenotypes of dao1-1. These results suggest that IAA oxidation plays an important role in IAA homeostasis during the whole life of Arabidopsis.