Abstract
The plant hormone auxin regulates numerous aspects of the plant life cycle. Auxin signalling is mediated by auxin response factors (ARFs) that dimerise with modulating Aux/IAA repressors. ARF3 (ETTIN or ETT) is atypical as it does not interact with Aux/IAA repressors. It is proposed to be a non-canonical auxin sensor, regulating diverse functions essential for development. This sensing ability relies on a unique C-terminal ETT specific domain (ES domain). Alignments of ETT orthologues across the angiosperm phylum revealed that the length and sequence identities of ES domains are poorly conserved. Computational predictors suggested the ES domains to be intrinsically disordered, explaining their tolerance of insertions, deletions and mutations during evolution. Nevertheless, five highly conserved short linear motifs were identified suggesting functional significance. High-throughput library screening identified an almost full-length soluble ES domain that did not bind auxin directly, but exhibited a dose-dependent response in a yeast two-hybrid system against the Arabidopsis INDEHISCENT (IND) transcription factor. Circular dichroism confirmed the domain was disordered. The identification and purification of this domain opens the way to the future characterisation of the ETT auxin-sensing mechanism in planta and an improved understanding of auxin-mediated regulation.
Highlights
The Arabidopsis genome encodes 23 auxin response factors (ARFs) with a conserved domain organisation: a DNA binding domain (DBD), located at the N terminus[4], and the PB1 (Phox and Bem1) domain at the C terminus that mediates binding to the Aux/IAA repressor and other ARF members[4,5]
The Auxin Response Factor ETT (ARF3) of A. thaliana is essential for correct female reproductive structure development[8,25]
For decades ETT has been proposed to act as an interpreter of auxin concentration in the female reproductive organ, but only recently has a molecular mechanism been proposed whereby auxin affects the interaction between ETT and protein partners, including transcription factors, leading to changes in the expression of downstream targets[8,25]
Summary
The Arabidopsis genome encodes 23 ARFs with a conserved domain organisation: a DNA binding domain (DBD), located at the N terminus[4], and the PB1 (Phox and Bem1) domain at the C terminus that mediates binding to the Aux/IAA repressor and other ARF members[4,5]. The results revealed that the ES domain is predominantly intrinsically unstructured, but bears striking motifs that are almost universally conserved, both in sequence and in linear organisation, across evolutionarily diverse ETT homologues The identification of these motifs allowed us to interpret A. thaliana phenotypic studies on ETT deletion and point mutants. A 27 kDa engineered form of the ES domain was identified by the high throughput ESPRIT method[10,11] that could be produced and purified at multi-milligram quantities, and yet exhibited identical auxin responses to the wild-type sequence in Y2H assay This opens the way for structural and biophysical studies into mechanisms of non-canonical auxin-mediated plant responses following identification of the molecular partners that interact with the conserved linear motifs in the ES domain
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