Analysis of the function and metabolism of indole-3-acetic acid conjugates in Arabidopsis thaliana

The auxin indole-3-acetic acid (IAA) is involved in virtually every aspect of plant development. Plants control auxin homeostasis through complex interactions between de novo synthesis, degradation, import, export, and conjugate synthesis and hydrolysis. A thorough knowledge of these pathways and their interactions is key to understanding auxin response and plant growth. I have used genetic and biochemical methods to better understand the function and metabolism of amide-linked conjugates of IAA. Conjugates may function as storage or transport forms of IAA, or may function independently of hydrolysis. Many IAA conjugates have auxin activity in bioassays, and Arabidopsis mutants with reduced sensitivity to exogenous IAA-L-amino acid conjugates have been identified. Some of these are defective in genes encoding conjugate hydrolases, whereas others are likely to be directly or indirectly involved in some other aspect of conjugate metabolism or transport. To better understand conjugate hydrolysis pathways, I have generated and screened an overexpression library to identify cDNAs that lead to conjugate resistance when overexpressed. From this library, I have identified one mutant that cosuppresses petH, resulting in a chlorotic phenotype, and several mutants resistant to IAA-Ala that result from mutations unlinked to T-DNA inserts. In addition, I have cloned the gene defective in iar4, an IAA-alanine-resistant mutant, and found that it encodes a protein similar to a mitochondrial pyruvate dehydrogenase E1alpha subunit. To better understand conjugate hydrolysis, I have characterized the enzyme activities of four members of the amidohydrolase family. I found that bacterially expressed GST fusions of ILR1, ILL2, and IAR3 hydrolyze certain IAA-amino acids with KM values in the muM range, suggesting they are physiologically relevant. In addition, mutant plant extracts show altered rates of conjugate hydrolysis, further supporting the hypothesis that these enzymes function in vivo to cleave IAA-amino acid conjugates. I have also examined the expression profiles of the amidohydrolase family and found their expression is overlapping but distinct. My findings suggest that the activity displayed by auxin conjugates is due to their hydrolysis by the amidohydrolases to yield free IAA, and that these processes are controlled both developmentally and spatially to regulate free IAA levels