Figure Legend: model for Auxin-Regulated Gene Expression. When levels of auxin are low in cells (top of figure), ARF activators are dimerized with Aux/IAA repressors on TGTCTC AuxREs in the promoters of auxin response genes. Because the repression domain in Aux/IAA proteins is dominant over the activation domain in the ARF protein, the auxin response genes are actively repressed. When auxin concentrations are increased, the Aux/IAA repressors are targeted through the ubiquitin-proteasome pathway for rapid degradation. This results in the loss of Aux/IAA repressors and the subsequent activation of the auxin response genes (bottom of figure). The ARF activator is shown with an N-terminal DNA-binding domain (DBD) in contact with TGTCTC AuxREs on an auxin response gene. The Q-rich (glutamine-rich) middle region of the ARF is the activation domain, and the two gold boxes in the C-terminus of the ARF represent the dimerization domain. This C-terminal dimerization domain is conserved in Aux/IAA proteins. The Aux/IAA proteins contain four conserved domains that are diagrammed as four boxes with the N-terminal green box representing the repression domain (RD), the blue box representing an instability domain, and the C-terminal gold boxes representing the dimerization domain. The central part of the figure represents the auxin-dependent degradation of the Aux/IAA repressors through the ubiquitin-proteasome pathway.

The plant hormone auxin, indole-3-acetic acid, plays a major role in regulating a plethora of plant growth and developmental responses. Many of these responses are brought about by auxin-induced changes in gene expression. At least in some cases, auxin regulates transcription of selected genes through auxin-responsive promoter elements (AuxREs) with the consensus sequence, TGTCTC. Two types of transcription factors are recruited to this element, Auxin Response Factors or ARFs and Aux/IAA proteins. Some ARFs are transcriptional activators with a glutamine-rich activation domain that bind directly to AuxREs. Aux/IAA proteins are repressors that do not directly bind to AuxREs, but are recruited to AuxREs by dimerizing with ARF activators that occupy AuxREs. The targeting of Aux/IAA proteins to AuxREs occurs when auxin concentrations are low and results in active repression of auxin response genes. When auxin concentrations are elevated, the Aux/IAA repressors are rapidly degraded by the ubiquitin-proteasome pathway, resulting in loss of the repressors and activation of the auxin response genes. This work identifies a portable repression domain (RD in the figure provided) in Aux/IAA proteins that is dominant over the activation domain found in ARF activators and provides a mechanism for repression of auxin response genes when auxin concentrations are low.

The repression domain identified in our studies is small, consisting of about 10 amino acids, and contains a conserved LxLxL motif, where L is leucine and x is one of several different amino acids. Because this repression domain is portable and dominant over at least some activation domains, the Aux/IAA repression domain can be transferred to other transcription factors and convert activators to repressors. Identification of the Aux/IAA repression domain also allows one to incapacitate this domain via site-directed mutagenesis and convert Aux/IAA repressors to activators.

Identification of a small repression domain in Aux/IAA proteins that is dominant over an ARF transcriptional activation domain provides a plausible explanation for the inactivation of auxin response genes when the amount of auxin in a cell is low or below a threshold. As long as Aux/IAA repressors are in contact with ARF activators on auxin response genes, the repressor wins, and auxin response genes are inactive or turned off. The auxin response genes are only activated when auxin levels become sufficiently high to trigger the destruction of the Aux/IAA repressors.

Our laboratory initially set out to demonstrate that auxin could rapidly regulate genes. After identifying such genes, we wanted to determine how these genes were regulated in terms of promoter elements and transcription factors. We used the promoters of genes that responded within minutes to applied auxin to identify the minimal element (i.e., the TGTCTC AuxRE) that could confer an auxin response. We then used the minimal AuxRE to identify the transcription factors that are bound to it. These transcription factors are unique to plants and named ARFs. Domains were identified in ARFs that are required for binding to TGTCTC AuxREs and for activation or repression. Subsequent studies from our laboratory and others showed that ARFs can dimerize with themselves and with Aux/IAA proteins through a conserved domain in both families of proteins. We showed that selected ARFs can activate specific genes in an auxin dose-dependent manner, and that Aux/IAA proteins can repress these same genes. We then carried out experiments to determine how Aux/IAA proteins function as repressors of auxin response genes.