Sunethra Dharmasiri

The F-box protein TIR1 is an auxin receptor

The plant hormone auxin regulates diverse aspects of plant growth and development. Recent studies indicate that auxin acts by promoting the degradation of the Aux/IAA transcriptional repressors through the action of the ubiquitin protein ligase SCFTIR1. The nature of the signalling cascade that leads to this effect is not known. However, recent studies indicate that the auxin receptor and other signalling components involved in this response are soluble factors. Using an in vitro pull-down assay, we demonstrate that the interaction between transport inhibitor response 1 (TIR1) and Aux/IAA proteins does not require stable modification of either protein. Instead auxin promotes the Aux/IAA–SCFTIR1 interaction by binding directly to SCFTIR1. We further show that the loss of TIR1 and three related F-box proteins eliminates saturable auxin binding in plant extracts. Finally, TIR1 synthesized in insect cells binds Aux/IAA proteins in an auxin-dependent manner. Together, these results indicate that TIR1 is an auxin receptor that mediates Aux/IAA degradation and auxin-regulated transcription.

Phosphate Availability Alters Lateral Root Development in Arabidopsis by Modulating Auxin Sensitivity via a Mechanism Involving the TIR1 Auxin Receptor

Lateral root development is an important morphogenetic process in plants, which allows the modulation root architecture and substantially determines the plants efficiency for water and nutrient uptake. Postembryonic root development is under the control of both endogenous developmental programs and environmental stimuli. Nutrient availability plays a major role among environmental signals that modulate root development. Phosphate (Pi) limitation is a constraint for plant growth in many natural and agricultural ecosystems. Plants possess Pi-sensing mechanisms that enable them to respond and adapt to conditions of limited Pi supply, including increased formation and growth of lateral roots. Root developmental modifications are mainly mediated by the plant hormone auxin. Recently we showed that the alteration of root system architecture under Pi-starvation may be mediated by modifications in auxin sensitivity in root cells via a mechanism involving the TIR1 auxin receptor. In this addendum, we provide additional novel evidence indicating that the low Pi pathway involves changes in cell cycle gene expression. It was found that Pi deprivation increases the expression of CDKA, E2Fa, Dp-E2F and CyCD3. In particular, E2Fa, Dp-E2F and CyCD3 genes were specifically upregulated by auxin in Pi-deprived Arabidopsis seedlings that were treated with the auxin transport inhibitor NPA, indicating that cell cycle modulation by low Pi signaling is independent of auxin transport and dependent on auxin sensitivity in the root.The survival of plants, as sessile organisms, depends on a series of postembryonic developmental events that determine the final architecture of plants and allow them to contend with a continuously changing environment. Modulation of cell differentiation and organ formation by environmental signals has not been studied in detail. Here, we report that alterations in the pattern of lateral root (LR) formation and emergence in response to phosphate (Pi) availability is mediated by changes in auxin sensitivity in Arabidopsis thaliana roots. These changes alter the expression of auxin-responsive genes and stimulate pericycle cells to proliferate. Modulation of auxin sensitivity by Pi was found to depend on the auxin receptor TRANSPORT INHIBITOR RESPONSE1 (TIR1) and the transcription factor AUXIN RESPONSE FACTOR19 (ARF19). We determined that Pi deprivation increases the expression of TIR1 in Arabidopsis seedlings and causes AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) auxin response repressors to be degraded. Based on our results, we propose a model in which auxin sensitivity is enhanced in Pi-deprived plants by an increased expression of TIR1, which accelerates the degradation of AUX/IAA proteins, thereby unshackling ARF transcription factors that activate/repress genes involved in LR formation and emergence.

Complex regulation of the TIR1/AFB family of auxin receptors.

Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.

AXR1-ECR1–Dependent Conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 Is Required for Auxin Response

Mutations in the AXR1 gene result in a reduction in auxin response and diverse defects in auxin-regulated growth and development. In a previous study, we showed that AXR1 forms a heterodimer with the ECR1 protein. This enzyme activates the ubiquitin-related protein RUB1 in vitro. Furthermore, we showed that the Skp1-Cul1/Cdc53-F-box (SCF) subunit AtCUL1 is modified by RUB1 in vivo. In this report, we demonstrate that the formation of RUB-AtCUL1 is dependent on AXR1 and ECR1 in vivo. The expression of AXR1 and ECR1 is restricted to zones of active cell division and cell elongation, consistent with their role in growth regulation. These results provide strong support for a model in which RUB conjugation of AtCUL1 affects the function of SCF E3s that are required for auxin response.

Role of the Arabidopsis RING-H2 protein RBX1 in RUB modification and SCF function

The ubiquitin-related protein RUB/Nedd8 is conjugated to members of the cullin family of proteins in plants, animals, and fungi. In Arabidopsis, the RUB conjugation pathway consists of a heterodimeric E1 (AXR1-ECR1) and a RUB-E2 called RCE1. The cullin CUL1 is a subunit in SCF-type ubiquitin protein ligases (E3s), including the SCFTIR1 complex, which is required for response to the plant hormone auxin. Our previous studies showed that conjugation of RUB to CUL1 is required for normal SCFTIR1 function. The RING-H2 finger protein RBX1 is a subunit of SCF complexes in fungi and animals. The function of RBX1 is to bind the ubiquitin-conjugating enzyme E2 and bring it into close proximity with the E3 substrate. We have identified two Arabidopsis genes encoding RING-H2 proteins related to human RBX1. Studies of one of these proteins indicate that, as in animals and fungi, Arabidopsis RBX1 is an SCF subunit. Reduced RBX1 levels result in severe defects in growth and development. Overexpression of RBX1 increases RUB modification of CUL1. This effect is associated with reduced auxin response and severe growth defects similar to those observed in axr1 mutants. As in the axr1 mutants, RBX1 overexpression stabilizes the SCFTIR1 substrate AXR2/IAA7. The RBX1 protein is a component of SCF complexes in Arabidopsis. In addition to its direct role in SCF E3 ligase activity, RBX1 promotes the RUB modification of CUL1 and probably functions as an E3 ligase in the RUB pathway. Hypermodification of CUL1 disrupts SCFTIR1 function, suggesting that cycles of RUB conjugation and removal are important for SCF activity.

Auxin Action in a Cell-Free System

The plant hormone auxin regulates diverse aspects of plant growth and development. Despite its importance, the mechanisms of auxin action remain poorly understood. In particular, the identities of the auxin receptor and other signaling proteins are unknown. Recent studies have shown that auxin acts by promoting the degradation of a family of transcriptional regulators called the Aux/IAA proteins. These proteins interact with another large family of plant-specific transcription factors called Auxin Response Factors (ARF) and negatively regulate their activity. Auxin stimulates Aux/IAA degradation by promoting the interaction between a ubiquitin protein ligase (E3) called SCF(TIR1) and the Aux/IAA protein. In this report, we demonstrate that auxin promotes the interaction between the Aux/IAA proteins and SCF(TIR1) in a soluble extract free of membranes, indicating that this auxin response is mediated by a soluble receptor. In addition, we show that the response is not dependent on protein phosphorylation or dephosphorylation but rather is prevented by an inhibitor of peptidyl-prolyl isomerases.

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCFTIR1. Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCFTIR1 substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.

AXR4 Is Required for Localization of the Auxin Influx Facilitator AUX1

The AUX1 and PIN auxin influx and efflux facilitators are key regulators of root growth and development. For root gravitropism to occur, AUX1 and PIN2 must transport auxin via the lateral root cap to elongating epidermal cells. Genetic studies suggest that AXR4 functions in the same pathway as AUX1. Here we show that AXR4 is a previously unidentified accessory protein of the endoplasmic reticulum (ER) that regulates localization of AUX1 but not of PIN proteins. Loss of AXR4 resulted in abnormal accumulation of AUX1 in the ER of epidermal cells, indicating that the axr4 agravitropic phenotype is caused by defective AUX1 trafficking in the root epidermis.

The Arabidopsis SUPPRESSOR OF AUXIN RESISTANCE Proteins Are Nucleoporins with an Important Role in Hormone Signaling and Development

Nucleocytoplasmic transport of macromolecules is regulated by a large multisubunit complex called the nuclear pore complex (NPC). Although this complex is well characterized in animals and fungi, there is relatively little information on the NPC in plants. The suppressor of auxin resistance1 (sar1) and sar3 mutants were identified as suppressors of the auxin-resistant1 (axr1) mutant. Molecular characterization of these genes reveals that they encode proteins with similarity to vertebrate nucleoporins, subunits of the NPC. Furthermore, a SAR3–green fluorescent protein fusion protein localizes to the nuclear membrane, indicating that SAR1 and SAR3 are Arabidopsis thaliana nucleoporins. Plants deficient in either protein exhibit pleiotropic growth defects that are further accentuated in sar1 sar3 double mutants. Both sar1 and sar3 mutations affect the localization of the transcriptional repressor AXR3/INDOLE ACETIC ACID17, providing a likely explanation for suppression of the phenotype conferred by axr1. In addition, sar1 sar3 plants accumulate polyadenylated RNA within the nucleus, indicating that SAR1 and SAR3 are required for mRNA export. Our results demonstrate the important role of the plant NPC in hormone signaling and development.

The role of regulated protein degradation in auxin response

Auxin-regulated gene expression is mediated by two families of transcription factors. The ARF proteins bind to a conserved DNA sequence called the AuxRE and activate transcription. The Aux/IAA proteins repress ARF function, presumably by forming dimers with ARF proteins. Recent genetic studies in Arabidopsis indicate that auxin regulates this system by promoting the ubiquitin-mediated degradation of the Aux/IAA proteins, thus permitting ARF function. Mutations in components of SCFTIR1, a ubiquitin protein ligase (E3) result in stabilization of Aux/IAA proteins and decreased auxin response. Further, recent biochemical experiments indicate that the Aux/IAA proteins bind SCFTIR1 in an auxin-dependent manner.