The AFB4 auxin receptor is a negative regulator of auxin signaling in seedlings

The plant hormone auxin is perceived by a family of F box proteins called the TIR1/auxin-signaling F box proteins (AFBs). Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling. In this report, we demonstrate a unique role for the AFB4 clade. Both AFB4 and AFB5 function as auxin receptors based on in vitro assays. However, unlike other members of the family, loss of AFB4 results in a range of growth defects that are consistent with auxin hypersensitivity, including increased hypocotyl and petiole elongation and increased numbers of lateral roots. Indeed, qRT-PCR experiments show that afb4-2 is hypersensitive to indole-3-acetic acid (IAA) in the hypocotyl, indicating that AFB4 is a negative regulator of auxin response. Furthermore, we show that AFB4 has a particularly important role in the response of seedlings to elevated temperature. Finally, we provide evidence that the AFB4 clade is the major target of the picloram family of auxinic herbicides. These results reveal a previously unknown aspect of auxin receptor function.     

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 SCF(TIR1). 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 SCF(TIR1) 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.     

Toyocamycin specifically inhibits auxin signaling mediated by SCF pathway

The auxins, plant hormones, play a crucial role in many aspects of plant development by regulating cell division, elongation and differentiation. Toyocamycin, a nucleoside-type antibiotic, was identified as auxin signaling inhibitor in a screen of microbial extracts for inhibition of the auxin-inducible reporter gene assay. Toyocamycin specifically inhibited auxin-responsive gene expression, but did not affect other hormone-inducible gene expression. Toyocamycin also blocked auxin-enhanced degradation of the Aux/IAA repressor modulated by the SCF(TIR1) ubiquitin-proteasome pathway without inhibiting proteolytic activity of proteasome. Furthermore, toyocamycin inhibited auxin-induced lateral root formation and epinastic growth of cotyledon in the Arabidopsis thaliana plant. This evidence suggested that toyocamycin would act on the ubiquitination process regulated by SCF(TIR1) machineries. To address the structural requirements for the specific activity of toyocamycin on auxin signaling, the structure-activity relationships of nine toyocamycin-related compounds, including sangivamycin and tubercidin, were investigated.     

Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.     

Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases

Selective protein degradation by the ubiquitin-proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin-ligases which act downstream of the ubiquitin-activating and -conjugating enzymes to identify specific substrates for ubiquitinylation. SCF-type ubiquitin-ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1-1 auxin response defect, we identified eta1/axr6-3, a recessive and temperature-sensitive mutation in the CUL1 core component of the SCF(TIR1) complex. The axr6-3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6-3 displays a pleiotropic phenotype with defects in numerous SCF-regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6-3 as a tool for identifying pathways likely to be regulated by SCF-mediated proteolysis and propose new roles for SCF regulation of the far-red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature-sensitive nature of the pleiotropically acting axr6-3 mutation opens promising possibilities for the identification and investigation of SCF-regulated pathways in Arabidopsis.