Legal Definition Auxin

The third natural auxin-like compound is PAA. It is currently the only phenyl derivative of AIA identified. As observed in vitro, active concentrations of PAAs are much higher than those of IAA (Fitzsimons, 1989; Small and Morris, 1990). PAA has been found in many different plant species at different concentration ranges (Wightman and Lighty, 1982) and PAA has been shown to interfere with the active flow of IAA in peas, thereby playing a role in the interaction of plant roots with soil microorganisms (Morris and Johnson, 1987; Slininger et al., 2004; Somers et al., 2005). In high concentrations, auxins are toxic and their activities mainly target broadleaf weeds via monocot species such as grasses and cereal crops. Because of these properties, many compounds with auxin-like activity have been developed and used as herbicides (Grossmann, 2010). In addition, synthetic auxins are used as active molecules in commercial solutions for horticulture because they promote the initiation of adventitious roots and the synchronization of flowering and fruit set. An essential feature of plant growth and development, facilitated by plant hormones, is adaptation to endogenous and environmental stimuli. In addition to the well-known role of abscisic acid, brassinosteroids, salicylic acid and gibberellins in these processes, auxin also plays an important role in plant adaptation. According to a strict definition, auxin IAA, a simple molecule consisting of an indole ring and a carboxylic acid side chain, is structurally similar to the amino acid tryptophan. In a broader sense, however, auxin includes compounds that can cause an analogous reaction such as AIA (Paque and Weijers, 2016). To date, three other naturally occurring endogenous auxins have been discovered in plants, namely indole-3-butyric acid (IBA), 4-chlorindole-3-acetic acid (4-Cl-IAA) and phenylacetic acid (PAA) (Fig. 1), which are active in bioassays (Simon and Petrášek, 2011).

Shortly after the structural characterization of IAA, a diverse group of synthetic compounds, such as naphthalene-1-acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and picloram (Fig. 1), were identified as auxin-like substances and have since been widely used as chemical tools in auxin biology studies and as plant growth regulators in agriculture and horticulture. Like TIR1/AFB, auxiliaries/LPNs can also be regulated post-transcriptionally. The microRNA identified miR847, which can be transcrisionally upregulated by auxin via TIR1, targets IAA28 mRNA (Wang and Guo, 2015). In this way, auxin is not only responsible for the degradation of the IAA28 protein via the SCFTIR1/AFB complex, but also for its mRNA. Another type of modification involves interaction aux/IAA with PHYTOCHROME A and phosphorylation by its protein kinase activity. It is suggested that such a modification could modulate nuclear Aux/IAA localization, metabolic stability, and the ability to interact with other Aux/IAA or ARF proteins (Colón-Carmona et al., 2000). Another type of Aux/IAA modification is the isomerization of proline residues localized in domain II, resulting in decreased sensitivity of Aux/IAA proteins to the SCFTIR1 complex (Dharmasiri et al., 2003). So far, phosphorylation and isomerization of Aux/IAA proteins have only been demonstrated in vitro, so the influence of these modifications on the auxin response in planta does not yet need to be properly evaluated. In addition, the oligomerization of auxiliaries/IAAs has been shown to play an important role in the effective suppression of FRA activity.

The affinity of TOPLESS or histone deacetylases increases in the presence of oligomers to ARF/IAA. This leads to a densification of the chromatin environment, leading to DNA condensation and prevention of auxin-sensitive gene expression (Korasick et al., 2014; Han et al., 2014). Although our knowledge of SKP2A signaling is much less detailed than what we know about TIR1/AFB, SKP2A appears to meet the basic requirements of an auxin receptor, although more detailed studies are needed to meet the classical criteria for a receptor [specific and saturable binding, specific physiological responses and limiting function of these responses (see also Jones and Sussman`s review, 2009)]. Phototropic motion (bending towards light) can be explained by cellular stretching by auxins. The concentration of auxin increases due to the migration of auxin to the shaded side. This results in more cellular stretching on the shaded side than on the light-exposed side. A second level of complexity arises from the fact that the components of the TIR1 aux/IAA signaling pathway typically comprise large families of proteins. In Arabidopsis, there are five TIR1 homologs, called AFB1-AFB5, all of which bind to auxin, but with different affinities (Calderon Villalobos et al., 2012). Aux/IAA proteins form a large family of 29 that share a common structure of four domains (called I-IV). Domain II (DII) is directly involved in the interaction with TIR1: the auxin molecule inserts into the auxin binding site of TIR1 and the DII domain binds both TIR1 and the auxin molecule to a lid-like structure that captures the auxin molecule between TIR1 and Aux/IAA (Tan et al., 2007).

Since aux/IAAs are directly part of the receptor-ligand interaction, they can be considered auxin co-receptors. This co-receptor concept is biologically relevant because the auxin interaction surfaces of the five TIR/AFB receptors and the 29 aux/IAA proteins are not strictly conserved, which could lead to different pairs of TIR/AFB-AUX/IAA coreceptors with different auxin affinities. Among the first generation of auxin antagonists, tert-butoxycarbonylaminohexyl-IAA (BH-IAA) (Fig. 3) is a potent molecule that has been used to confirm that the SCFTIR1 aux/IAA signaling pathway is conserved between lower and upper terrestrial plants (Hayashi et al., 2008). In order to further improve the binding affinity of auxin antagonists for TIR1 to a much higher level than that of endogenous IAA, an approach based on the crystal structure of the TIR1-BH-IAA complex and virtual in silico screening of TIR1 ligands from chemical banks was chosen. The first virtual screening identified α-(phenyl-2-oxo)-IAA (PEO-IAA) (Fig. 3) as a very potent candidate compound. PEO-IAA phenyl ring methylation significantly increased auxin binding inhibitor activity, resulting in the synthesis of the optimized final compound auxinol (α-(2,4-dimethylphenyl-2-oxo)-IAA) (Fig. 3) (Hayashi et al., 2012). Analysis of the molecular docking of auxinol and PEO-IAA predicts that the phenyl ring of the two ligands effectively prevents the DII motif from accessing the Aux/IAA protein. In accordance with this intended mechanism of action, auxinols and PEO-IAA have been shown to competitively inhibit various auxin reactions in planta by blocking the formation of the TIR1-IAA-Aux/IAA complex and stabilizing aux/IAA repressors (Hayashi et al., 2012).

Today, auxinol and PEO-IAA are widely used to reversibly block the action of auxin, providing novel and powerful tools for chemical-biological analysis of auxin-regulated processes in various plant species (Tatsuki et al., 2013; Procko et al., 2014; Fendrych et al., 2016). The basic structure of all members of the TIR1/AFB family consists of a region of 18 leucine-rich repeats (LRR) at the N end and an F-box domain at the C end. The crystal structure of TIR1 in complex with the SCF complex protein ARABIDOPSIS SKP1 HOMOLOGUE (ASK1), a small peptide aux/IAA of IAA7, and auxin showed that the auxin binding pocket is formed by the LRR TIR1 domain, while the TIR1 F-box domain interacts with ASK1 (Tan et al., 2007).