Characterization of Aux/IAA Protein Degradation in Higher Plants

University Of California-Davis, Davis CA

Investigators

Abstract

The regulation of proteolysis is an emerging paradigm in cellular and organismal regulation. Multiple pathways have been demonstrated to be regulated through the specific and controlled degradation of both regulatory and downstream proteins. In higher plants, multiple aspects of plant growth and development require auxin, of which indole-3-acetic acid (IAA) is the most abundant naturally occurring biologically active member. Auxin regulates cell division, cell elongation, cell differentiation, and it plays an important role in multiple organismal processes such as lateral root initiation, apical dominance, and tropic responses. Molecular and biochemical studies have identified the family of Aux/IAA proteins as essential players in the auxin signal transduction pathway that alters gene expression in plants. In addition to the activity of the Aux/IAA proteins (which remains poorly characterized), their rapid degradation has emerged as an integral part of their biological function. The mechanism and role of this rapid proteolysis is the focus of this study. Based on work in other systems demonstrating that the cis-acting requirement for proteolysis is represented by a small region that can target other proteins for degradation, the rapid degradation of a fusion of Aux/IAA amino acids to the marker enzyme firefly luciferase was demonstrated directly in transgenic Arabidopsis plants. The nature of absolutely required Aux/IAA amino acids were defined, however, flanking sequences appear to affect proteolysis in a way that was not completely understood. Experiments proposed here will define the role of these sequences. Point mutations and deletions of Aux/IAA sequences will be made, fused to LUC and the fusion protein half-life determined in transgenic plants. To determine whether all Aux/IAA proteins have the same half-life, the ability of additional Aux/IAA proteins to target LUC for degradation will be determined. An Aux/IAA protein lacking the required conserved amino acids will tested for in vivo degradation as a LUC fusion. If not rapidly degraded, this protein will serve as the starting point to determine the sufficiency of the Aux/IAA degradation signal. Application of exogenous auxin alters the rate of Aux/IAA proteolysis, providing for the first time experimental evidence for a proposed mechanism for auxin-regulated gene expression. Increased loss of Aux/IAA proteins by faster proteolysis correlated with induction of transcription of genes containing auxin-response elements in their regulatory regions. Experiments will be performed to test whether alterations in endogenous auxin alter Aux/IAA proteolysis through the construction of transgenic plants with inducible expression of enzymes that alter IAA steady state levels. The above studies will be complemented by a genetic approach to isolate mutants defective in Aux/IAA::LUC degradation. Additional screens, including an activation tagging screen, will be initiated. The effect on Aux/IAA::LUC proteolysis of previously described mutants altered in auxin responses will also be determined to identify genes important for Aux/IAA proteolysis. The results from the proposed work will be highly significant given the importance of the regulation of Aux/IAA proteolysis in auxin signaling and the importance of auxin in regulating plant growth and development. Aux/IAA proteolysis will serve as a model by which other cellular pathways may be regulated, providing methodogy, reagents, and experimental approaches useful in other systems. Sequences defined in these studies may be used in other contexts to control the intracellular level of a protein.