Auxinole

Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole

Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.

Rational design of an auxin antagonist of the SCF(TIR1) auxin receptor complex

The plant hormone auxin is a master regulator of plant growth and development. By regulating rates of cell division and elongation and triggering specific patterning events, indole 3-acetic acid (IAA) regulates almost every aspect of plant development. The perception of auxin involves the formation of a ternary complex consisting of an F-box protein of the TIR1/AFB family of auxin receptors, the auxin molecule, and a member the Aux/IAA family of co-repressor proteins. In this study, we identified a potent auxin antagonist, α-(phenylethyl-2-oxo)-IAA, as a lead compound for TIR1/AFB receptors by in silico virtual screening. This molecule was used as the basis for the development of a more potent TIR1 antagonist, auxinole (α-[2,4-dimethylphenylethyl-2-oxo]-IAA), using a structure-based drug design approach. Auxinole binds TIR1 to block the formation of the TIR1-IAA-Aux/IAA complex and so inhibits auxin-responsive gene expression. Molecular docking analysis indicates that the phenyl ring in auxinole would strongly interact with Phe82 of TIR1, a residue that is crucial for Aux/IAA recognition. Consistent with this predicted mode of action, auxinole competitively inhibits various auxin responses in planta. Additionally, auxinole blocks auxin responses of the moss Physcomitrella patens, suggesting activity over a broad range of species. Our works not only substantiates the utility of chemical tools for plant biology but also demonstrates a new class of small molecule inhibitor of protein-protein interactions common to mechanisms of perception of other plant hormones, such as jasmonate, gibberellin, and abscisic acid.

AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling

Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca²⁺ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA^- triggered calcium signals, are blocked by treatment with the SCF^TIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCF^TIR1/AFB receptor and the CNGC14 Ca²⁺ channel, mediate fast auxin signaling in roots.

The role of auxin in developmentally regulated programmed cell death in lace plant

Premise: Lace plant (Aponogeton madagascariensis) leaves are remodeled via developmental programmed cell death (PCD) to produce perforations located equidistantly between longitudinal and transverse veins. Auxin has been implicated in other developmental PCD processes in plants; however, the role of auxin in perforation formation in lace plant is unknown. Here the role of auxin in developmental PCD in lace plant was studied using two auxin inhibitors N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, and auxinole, a potent auxin antagonist.
Methods: Sterile cultures of lace plants were propagated and treated with NPA or auxinole. Leaf length, leaf width, and number of perforations were then analyzed. Vein patterning and perforation area were further examined in NPA-treated plants. Downstream PCD transduction events were investigated via spectrophotometric assays, histochemical staining, and immuno-probing.
Results: Lace plants treated with NPA or auxinole produced leaves with fewer perforations compared to their respective controls. Although NPA treatment was insufficient to completely alter vein patterning, NPA-treated leaves did have significantly more atypical areoles compared to control leaves. Events involved in perforation formation in lace plant leaves were altered following treatment with NPA, including anthocyanin production, reactive oxygen species (ROS) accumulation, and the release of mitochondrial cytochrome c.
Conclusions: Our results indicated that inhibition of auxin signaling disrupts several downstream features of the lace plant PCD signaling cascade and results in fewer or no perforations. Therefore, we concluded that auxin signaling is important for developmentally regulated PCD in lace plant leaves.

Protoplast Swelling and Hypocotyl Growth Depend on Different Auxin Signaling Pathways

Members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known auxin receptors. To analyze the possible receptor function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor currently under debate, we performed different approaches. We performed a pharmacological approach using α-(2,4-dimethylphenylethyl-2-oxo)-indole-3-acetic acid (auxinole), α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), and 5-fluoroindole-3-acetic acid (5-F-IAA) to discriminate between ABP1- and TIR1/AFB-mediated processes in Arabidopsis (Arabidopsis thaliana). We used a peptide of the carboxyl-terminal region of AtABP1 as a tool. We performed mutant analysis with the null alleles of ABP1, abp1-c1 and abp1-TD1, and the TILLING mutant abp1-5 We employed Coimbra, an accession that exhibits an amino acid exchange in the auxin-binding domain of ABP1. We measured either volume changes of single hypocotyl protoplasts or hypocotyl growth, both at high temporal resolution. 5-F-IAA selectively activated the TIR1/AFB pathway but did not induce protoplast swelling; instead, it showed auxin activity in the hypocotyl growth test. In contrast, PEO-IAA induced an auxin-like swelling response but no hypocotyl growth. The carboxyl-terminal peptide of AtABP1 induced an auxin-like swelling response. In the ABP1-related mutants and Coimbra, no auxin-induced protoplast swelling occurred. ABP1 seems to be involved in mediating rapid auxin-induced protoplast swelling, but it is not involved in the control of rapid auxin-induced growth.