Refining the nuclear auxin response pathway through structural biology

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Highlights

  • Structural biology studies refined the auxin response pathway.

  • The TIR1 E3 ubiquitin ligase structure reveals the auxin perception mechanism.

  • ARF DNA binding domain structures establish the ‘molecular calipers’ mechanism.

  • ARF and Aux/IAA PB1 interaction domain structures increase pathway complexity.

Auxin is a key regulator of plant growth and development. Classical molecular and genetic techniques employed over the past 20 years identified the major players in auxin-mediated gene expression and suggest a canonical auxin response pathway. In recent years, structural and biophysical studies clarified the molecular details of auxin perception, the recognition of DNA by auxin transcription factors, and the interaction of auxin transcription factors with repressor proteins. These studies refine the auxin signal transduction model and raise new questions that increase the complexity of auxin signaling.

Introduction

The phytohormone auxin (indole-3-acetic acid, IAA) is a master regulator of plant growth and development through control of cell division and expansion [1]. Because auxin potently impacts growth and development, auxin regulation must be precise. A variety of auxin regulatory strategies, ranging from biosynthesis and metabolism control [2, 3, 4] to transport and localization within the plant [5, 6], influence plant growth and development. Ultimately, auxin sensing triggers the myriad of gene expression changes required for plant growth and development.

Nuclear auxin response pathway components were identified using molecular and genetic approaches to uncover three major families of proteins that intimately link hormone perception and gene expression. Discovery of these protein families  for example, the auxin binding F-box proteins (TIR1; TRANSPORT INHIBITOR RESPONSE 1 and AFB1–5; AUXIN SIGNALING F-BOX PROTEINS 1–5), the AUXIN RESPONSE FACTOR (ARF) transcription factors, and AUXIN/INDOLE 3-ACETIC ACID INDUCIBLE (Aux/IAA) repressor proteins  employed a combination of screens and phylogenetics [7, 8, 9, 10, 11].

Integration of these proteins into a pathway from auxin perception to gene expression occurred through a series of creative studies. Protoplast and seedling-based assays, established the canonical ‘domain’ organization of the ARF transcription factors as an N-terminal DNA-binding domain (DBD), a middle region (MR) conferring either activation or repression properties, and a C-terminal region containing two sequence motifs (III/IV) [12] (Figure 1a). Subsequent studies determined that the III/IV motif of ARF and Aux/IAA proteins mediates ARF•ARF, Aux/IAA•Aux/IAA, and ARF•Aux/IAA interactions [8, 13]; that the Aux/IAA motif I facilitates interaction with TOPLESS (TPL) co-repressors [14]; and that the Aux/IAA motif II contains a degron that controls protein stability [15]. These investigations also established Aux/IAA proteins as ARF repressors [16] and defined two ARF subfamilies  positive and negative transcriptional regulators [12]. Further, molecular biology-focused studies suggested TIR1 as an auxin receptor [17, 18].

Various features of these three protein families led to a general model for plant auxin responses (Figure 1b). Under low auxin, Aux/IAA proteins repress ARF-mediated auxin-responsive gene transcription. Upon increased auxin, IAA binds an auxin-perceiving F-box protein, permitting Aux/IAA interaction. TIR1/AFB•auxin•Aux/IAA complex formation leads to Aux/IAA ubiquitylation and degradation and frees ARF proteins to regulate auxin-responsive gene expression [19].

A series of recent structural biology studies revealed salient features that guide the molecular interplay between IAA and auxin signaling components. This review summarizes current structural biology contributions to the establishment, dissection, and refinement of the auxin response pathway.

Section snippets

Structural identification of auxin signal perception by SCFTIR1/AFB

The TIR1/AFB F-box protein family provides a mechanism for auxin perception and mediates Aux/IAA protein ubiquitylation for proteasomal degradation [11, 20, 21]. After initial studies identified TIR1 as an auxin receptor [17, 18], the landmark structural study by Tan et al. [22••] introduced a new mechanism for auxin perception and Aux/IAA degradation (Figure 2). The TIR1•auxin•Aux/IAA complex X-ray crystal structure revealed how auxin binds to TIR1 to mediate interaction with the IAA7 degron

Mechanistic clues of auxin responsive gene transcription through binding of AuxREs

ARF proteins are central players in the nuclear auxin response pathway. Initially discovered in a yeast one-hybrid screen for proteins that bind the canonical auxin response element (AuxRE) [7, 30], these proteins modulate auxin-responsive gene transcription. The Arabidopsis genome encodes 22 ARF proteins with a three-domain architecture (Figure 1a), consisting of an N-terminal B3 type DBD, middle region, and interaction domain. B3 domains are plant-specific DBD [31]. The ARF MR can be enriched

ARF and Aux/IAA PB1 domain structures introduce a multimerization option

ARF and Aux/IAA proteins interact through two C-terminal regions of sequence homology (i.e., sequence motifs III and IV) [8]. X-ray crystal structures of the C-terminal regions of Arabidopsis ARF7 [39••] and ARF5 [40••] demonstrate that region III/IV adopts a type I/II Phox/Bem1p (PB1) domain structure (Figure 4a), as suggested by bioinformatic analysis [41]. Likewise, NMR structures of the PB1 domains of Arabidopsis IAA17 [42••] and Pisum sativum IAA4 [43••] confirm a similar architecture in

Conclusions

Structural biology provided new insights that define and refine the auxin response pathway. The first views of the TIR1•auxin•Aux/IAA complex established the ‘molecular glue’ mechanism for auxin perception; ARF DBD structural analyses revealed a molecular basis for AuxRE recognition; and ARF and Aux/IAA PB1 domain structures led to insight into how protein interactions attenuate auxin responses in plants and raises the possibility of ARF and/or Aux/IAA multimerization. Together, these studies

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Support from the National Science Foundation Graduate Research Fellowship Program (2011101911 to D.A.K.), the United States Department of Agriculture – National Institute of Food and Agriculture Fellowship Program (MOW-2014-01877 to D.A.K.), the National Institutes of Health (R00 GM089987-03 to L.C.S.), and the National Science Foundation (MCB-1157771 to J.M.J. and IOS-1453750 to L.C.S.) is acknowledged.

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