Genetic analysis of IAA-conjugate sensitivity in Arabidopsis thaliana: Hydrolysis, transcriptional regulation, and metal homeostasis
Rampey, Rebekah A.
Doctor of Philosophy
Auxins are hormones important for numerous processes throughout plant development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. To examine the in vivo importance of auxin-conjugate hydrolysis, I generated a triple hydrolase mutant, ilr1 iar3 ill2 , which is deficient in three of these hydrolases. I compared root and hypocotyl growth of the single, double and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis. The regulation of IAA-conjugate hydrolase gene expression remains unknown. I have characterized the IAA-Leu-resistant mutant, ilr3-1, and cloned the defective gene, which encodes a basic helix-loop-helix leucine zipper transcription factor. ILR3 may modulate IAA-conjugate hydrolysis by regulating hydrolase expression or activity. Previous work on the IAA-Ala-resistant mutant iar1, which is defective in a predicted membrane protein similar to metal transporters, suggests a link between metal homeostasis and IAA-conjugate sensitivity. To gain insight into the function of IAR1, I conducted a genetic modifier screen to isolate second-site mutations that restore IAA-conjugate sensitivity to iar1. Here, I characterize one such mutant, mtpc2-1, and identify the gene defective as encoding a metal transport protein. The work described in this thesis suggests roles for multiple components in IAA-conjugate sensitivity in Arabidopsis, including conjugate hydrolysis, transcriptional regulation of relevant genes, and metal homeostasis.