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Transcriptional Responses to
the Auxin Hormone
Dolf Weijers1
and Doris Wagner2
1
Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen,
The Netherlands; email: dolf.weijers@wur.nl
2
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104;
email: wagnerdo@sas.upenn.edu
Annu. Rev. Plant Biol. 2016. 67:539–74
First published online as a Review in Advance on
February 22, 2016
The Annual Review of Plant Biology is online at
plant.annualreviews.org
This article’s doi:
10.1146/annurev-arplant-043015-112122
Copyright c 2016 by Annual Reviews.
All rights reserved
Keywords
auxin, gene expression, ARF, chromatin, specificity, development
Abstract
Auxin is arguably the most important signaling molecule in plants, and the
last few decades have seen remarkable breakthroughs in understanding its
production, transport, and perception. Recent investigations have focused
on transcriptional responses to auxin, providing novel insight into the functions
of the domains of key transcription regulators in responses to the
hormonal cue and prominently implicating chromatin regulation in these
responses. In addition, studies are beginning to identify direct targets of
the auxin-responsive transcription factors that underlie auxin modulation
of development. Mechanisms to tune the response to different auxin levels
are emerging, as are first insights into how this single hormone can trigger
diverse responses. Key unanswered questions center on the mechanism for
auxin-directed transcriptional repression and the identity of additional determinants
of auxin response specificity. Much of what has been learned in
model plants holds true in other species, including the earliest land plants.
539
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ANNUAL
REVIEWS Further
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TRANSPORT
INHIBITOR
RESISTANT
1/AUXIN
SIGNALING
F-BOX (TIR1/AFB)
proteins: nuclear
F-box proteins
that—together with
Aux/IAA
proteins—form the
auxin receptor
AUXIN/INDOLE-3ACETIC
ACID
(Aux/IAA) proteins:
small nuclear proteins
that physically interact
with ARFs and prevent
them from regulating
transcription; they also
serve as part of the
auxin receptor
AUXIN RESPONSE
FACTORs (ARFs):
sequence-specific
DNA-binding proteins
that trigger
transcriptional changes
in responses to auxin
Contents
THE PLAYERS: CORE COMPONENTS THAT TRANSLATE AUXIN
SENSING TO TRANSCRIPTIONAL RESPONSES. . . . . . . . . . . . . . . . . . . . . . . . . . . 540
QUANTITATIVE CONTROL OF TRANSCRIPTIONAL OUTPUT
IN RESPONSE TO AUXIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
Aux/IAA Degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545
Transcriptional Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
Posttranscriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Posttranslational Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
Protein Interactions and Oligomerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
GENERATING SPECIFICITY IN AUXIN RESPONSES. . . . . . . . . . . . . . . . . . . . . . . . . 549
Protein-DNA Interactions Select Auxin-Responsive Genes . . . . . . . . . . . . . . . . . . . . . . . 550
Cofactors Can Modulate Auxin Output . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Chromatin-Level Control in Auxin Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
TRANSCRIPTIONAL CONTROL OF AUXIN-DEPENDENT PROCESSES. . . . 552
Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 552
Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 555
Organogenesis from the Reproductive Shoot Apex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
Auxin Response During Gynoecium, Ovule, and Pollen Development. . . . . . . . . . . . . 559
THE EVOLUTION OF THE AUXIN RESPONSE PATHWAY. . . . . . . . . . . . . . . . . . 560
This review examines recent advances in our understanding of how auxin sensing triggers changes
in transcription or in cellular properties during plant development. Current challenges include
identifying the mechanism by which auxin gradients are read and interpreted to enable to cell type–
specific transcriptional or cellular responses. The individual contributions of the large number of
paralogous factors that encode the key components of the auxin response pathway in flowering
plants also remain to be determined. In addition, very few target genes are known to be directly
controlled by auxin-responsive transcription factors. Finally, the cascade of events required for
auxin-dependent changes in gene activation and repression needs to be elucidated.
THE PLAYERS: CORE COMPONENTS THAT TRANSLATE AUXIN
SENSING TO TRANSCRIPTIONAL RESPONSES
The path from auxin signal perception to altered gene expression is short in both the physical and
genetic senses. The key components of this pathway are the TRANSPORT INHIBITOR RESISTANT
1/AUXIN SIGNALING F-BOX (TIR1/AFB) F-box proteins, the AUXIN/INDOLE-3ACETIC
ACID (Aux/IAA) transcriptional coregulators, and sequence-specific binding proteins
called AUXIN RESPONSE FACTORs (ARFs). A coreceptor comprising a TIR1/AFB F-box
protein and an Aux/IAA transcriptional coregulator senses auxin (21). Auxin promotes the interaction
between TIR1/AFB and Aux/IAA, thereby triggering ubiquitin-mediated degradation of
the Aux/IAA proteins via the proteasome (72, 234). Aux/IAA proteins generally act as corepressors
to prevent auxin-responsive transcription (140, 196, 197, 203). Arabidopsis has 6 and 29 paralogous
members in the TIR1/AFB and Aux/IAA families, respectively.
TIR1/AFB proteins are incorporated into a four-subunit SCFTIR1/AFB
complex. Both this complex
and the small Aux/IAA proteins are localized in the nucleus (2). All TIR1/AFB proteins bind
auxin (46, 143). Four TIR1/AFB family members have been shown to promote auxin responses,
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Auxin response
elements (AuxREs):
cis-elements bound by
ARF proteins
and mutants in these factors are either subtly auxin resistant as single mutants (tir1, afb2, and afb3)
or strongly resistant as higher-order mutants (afb1) (49, 143). The tir1/afb mutants also display
morphological defects consistent with a role in auxin perception. Mutants in other components
of the SCFTIR1/AFB
ubiquitin ligase complex, such as ARABIDOPSIS SKP1 HOMOLOGUE
(ASK1), CULLIN 1 (CUL1), or RING-BOX 1 (RBX1), also cause auxin resistance (67, 70–72,
86, 124). Members of the TIR1/AFB family have an N-terminal leucine-rich-repeat region and a
C-terminal F-box domain (Figure 1a). AFB4 and AFB5 have additional protein domains and bind
auxin analogs differently than do TIR1 and AFB1–3 (21). The crystal structure of TIR1-ASK1
in a complex with auxin and a small Aux/IAA peptide has been solved (193), revealing that the
leucine-rich-repeat domain of TIR1/AFB contains the auxin-binding pocket, whereas the F-box
domain contacts ASK1. The Aux/IAA peptide was also in contact with the leucine-rich-repeat domain
at the auxin-binding site. These results suggest that auxin stabilizes the interaction between
TIR1/ASK and the Aux/IAA proteins (reviewed in 144, 172). The structure of TIR1 with fulllength
Aux/IAA has not been solved. The TIR1/AFB proteins also contact the CUL1 subunit of
the SCFTIR1/AFB
complex via the F-box domain, which is linked to autocatalytic degradation (233).
Aux/IAA proteins do not have DNA-binding motifs and are instead recruited to genomic
regions by ARF proteins, with whom they physically interact via shared C-terminal domains
(196, 228) (Figure 1b). Aux/IAA proteins in general have one or two N-terminal ETHYLENERESPONSIVE
ELEMENT BINDING FACTOR–ASSOCIATED REPRESSOR (EAR) or
EAR-like repressor motifs (domain 1); a central region that is required for the TIR1/AFB interaction
and hence for degradation (the degron, or domain 2); and a C-terminal Phox and Bem 1 (PB1)
protein-protein interaction domain that mediates both homo- and heterodimerization (reviewed
in 75) (Figure 1b). Two recent studies demonstrated that the multimerization of Aux/IAA proteins
in a head-tail configuration is mediated by the PB1 domain (50, 100). Earlier studies had revealed
strikingly different auxin sensitivities for different Aux/IAA proteins with half-lives from minutes
to hours (52, 72, 139, 222, 234). An elegant follow-up study showed that this translates into different
auxin-binding affinities of the coreceptor (Kd ranging from 10 to 300 nM) (21) and concluded
that the auxin sensitivity of the coreceptor is determined largely by the Aux/IAA moiety. However,
the TIR1/AFB moiety also contributes to the auxin sensitivity of the coreceptor (21, 143).
Individual Aux/IAA proteins differ significantly in the presence and conservation of domains
important for auxin sensitivity/protein stability and association with TIR1 (52). Most important
in this regard is the degron (domain 2) and a conserved pair of amino acids [lysine and arginine
(KR)] between domains 1 and 2 (Figure 1b). The presence of a canonical degron and the KR motif
enhances the affinity of the coreceptor for auxin and causes lower stability (degradation at a lower
auxin concentration) of the Aux/IAA protein (21, 52, 82, 125, 139, 154, 222). Some Aux/IAAs have
variant degrons or entirely lack this domain, which leads to reduced sensitivity or insensitivity to
auxin, respectively (21, 52, 125). Mutations in the degron render Aux/IAA proteins insensitive to
auxin (reviewed in 156), presumably because the core residues in the degron directly interact with
the TIR1-auxin complex (193). Aux/IAAs that carry a lysine-glutamine (KQ) motif or have no
KR motif also exhibit decreased auxin sensitivity (21, 52, 125). The presence of polar amino acids
downstream of the degron enhances TIR1/AFB interaction, and their mutation leads to gain-offunction
phenotypes of Aux/IAA proteins that are similar to but less severe than mutations in the
degron motif (125). The degron has been engineered into enhanced sensitivity auxin reporters
(20, 107) and has been employed as a regulatory module in metazoans (88, 132).
Key for the transcriptional response to auxin are sequence-specific transcriptional regulators of
the ARF family. ARF proteins bind to so-called auxin response elements (AuxREs), cis-regulatory
sequences that at their core contain a TGTC motif that is sufficient to recruit ARF proteins
(195, 199, 201). Recent elegant structural analyses as well as protein array data have indicated
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F-box LRR
Tethering to
SCF complex
Auxin and Aux/IAA
interaction
PB1DI DII
ARF and Aux/IAA
interaction
EAR
domain
(Δ in IAA33)
TPL
Degron: GWPP(V/I)
(Δ in IAA20 and -30–34)
TIR1-auxin interaction
PB1MR MRB3DD DD
Aux/IAA & ARF
interaction
(Δ in ARF3 and -17)
Transcriptional regulation
EAR domain
(in ARF2, -9,
-18, and -19)
Dimerization
DNA-binding
Q rich
(class A ARFs)
P, S, and T rich
(class B/C ARFs)
(ARF5)
TPL
SYD/BRM
ARFc
Aux/IAAb
TIR1/AFBa
Figure 1
Domain architecture of central components of auxin-dependent gene regulation. Auxin responses are
mediated by interactions (arrows) between three core components: (a) TIR1/AFB auxin receptors,
(b) Aux/IAA transcriptional repressors, and (c) ARF transcription factors. TIR1/AFB proteins contain an
F-box domain for tethering to the other subunits in the SCF E3 ubiquitin ligase complex and a
leucine-rich-repeat (LRR) domain that carries the auxin-binding pocket and Aux/IAA contact site. Aux/IAA
proteins consist of domain 1 (DI, missing in IAA33), which harbors an EAR motif that mediates interaction
with TPL; domain 2 (DII, missing in IAA20 and -30–34), which carries the degron [the conserved amino
acid sequence GWPP(V/I), which acts as the contact site with TIR1/AFB and auxin]; and a PB1 domain,
which mediates oligomerization and Aux/IAA-ARF heterodimerization. ARFs have an N-terminal B3
DNA-binding domain flanked on either side by a dimerization domain (DD), followed by a middle region
(MR) that mediates transcriptional regulation. This domain can contain an EAR motif (in ARF2, -9, -18, and
-19) for interaction with TPL; it is glutamine (Q) rich in class A ARFs but proline (P), serine (S), and
threonine (T) rich in class B and C ARFs. In ARF5, this domain mediates the interaction with SYD and
BRM. At their C termini, ARFs (with the exception of ARF3 and -17) have a PB1 domain for
oligomerization and Aux/IAA-ARF heterodimerization. Protein abbreviations: ARF, AUXIN RESPONSE
FACTOR; Aux/IAA, AUXIN/INDOLE-3-ACETIC ACID; BRM, BRAHMA; EAR, ETHYLENERESPONSIVE
ELEMENT BINDING FACTOR–ASSOCIATED REPRESSOR; PB1, Phox and Bem 1;
SYD, SPLAYED; TIR1/AFB, TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING
F-BOX; TPL, TOPLESS.
that diverse Arabidopsis ARFs (ARF1, -3, and -5) preferentially bind the larger TGTCGG motif
(17, 61). ARF proteins can be grouped into three classes from the early land plants onward (58,
59, 95) (Figure 1c). Class A comprises ARFs with a glutamine (Q)–rich middle region that are
classified as transcriptional activators based on transient gene expression assays in protoplasts
(200). The Q-rich domain is present in all class A ARFs (58, 95). Recently, characterization of an
allelic series of monopteros (mp) mutant alleles of ARF5 in the Arabidopsis Columbia ecotype has
highlighted the importance of this domain (133). The remaining ARFs are classified as repressors
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TOPLESS (TPL):
a corepressor protein
that interacts with
EAR domain–
containing proteins
(including Aux/IAA
proteins), forms
tetramers, and recruits
chromatin regulators
such as HDACs
Histone deacetylases
(HDACs): enzymes
that remove acetyl
groups from lysines on
histones and form a
compact chromatin
state refractory to
transcription
SWITCH/
SUCROSE NON-
FERMENTING
(SWI/SNF)
chromatin-
remodeling
complexes: large
(2-MDa) complexes
that utilize energy
derived from ATP
hydrolysis to alter the
accessibility of
genomic DNA in the
context of chromatin
based on the same protoplast assay or sequence homology (195, 200) and can be divided into the
microRNA 160 (miR160)–targeted ARFs (class C) and the remaining ARFs (class B) (Figure 1c).
Structural analyses of the ARF DNA-binding region revealed that the B3 DNA-binding domain is
embedded in an ARF dimerization domain and that ARF proteins preferentially bind to inverted
AuxRE repeats (17). Most but not all ARFs have a C-terminal PB1 domain (Figure 1c), which is
important for the physical interactions between ARFs and Aux/IAA proteins (100, 129). Aux/IAA
proteins inhibit the transcriptional activity of ARFs specifically under low-auxin conditions (197).
The PB1 domain may also contribute to ARF-ARF dimerization (75, 100, 129).
How ARFs execute their roles in gene repression is not well understood. Although many class
B and C ARFs have PB1 domains, most appear to not interact very strongly with Aux/IAA proteins
(146, 206). Richter et al. (162) have suggested that class B and C ARFs may interfere with the
activity of class A ARFs in an auxin-independent fashion, for example, by competing for DNAbinding
sites or blocking activating ARF activity via heterodimerization. However, genetic studies
have linked class B and C ARFs to auxin-regulated processes (131, 180, 181), whereas transient
transcription assays have suggested an Aux/IAA-independent mechanism for auxin-regulated gene
expression also for class A ARFs (211). Several class B (repressive) ARFs (ARF2, -9, and -18) and a
single class A ARF (ARF19) have an EAR domain, implicating them in recruitment of corepressor
complexes (23). Because studies have also suggested that class A ARFs repress certain target genes
(235, 237), there is at present no satisfying model for auxin-dependent gene repression.
Aux/IAA inhibits activating ARFs bound at their target loci by recruitment of corepressor
complexes. The EAR repressor motif in domain 1 of the Aux/IAA proteins physically interacts
with and recruits Tup1/Groucho/TLE family proteins called TOPLESS (TPL) or TOPLESS
RELATED (TPR) (23, 96, 116, 192) (Figure 2). The cocrystal between TPL and the Aux/IAA
EAR domain has recently been solved (96), showing that the interaction requires the so-called
CTLH region at the TPL N terminus. The EAR motif–interacting region of TPL forms a
tetramer that interacts more strongly with oligomerized EAR domain–containing partners, which
may represent one biological role for the observed Aux/IAA protein oligomerization (100). Other
components may also be needed for repression. In addition to its important function in preventing
unlicensed gene expression in the auxin pathway, TPL also interacts with many other types of
EAR domain–containing transcriptional repressors in diverse developmental and stress pathways
(23, 135, 167).
Repression of auxin response gene expression in low auxin further requires histone deacetylases
(HDACs) such as HDA19. Loss of HDA19 activity partially rescues the phenotypes associated
with gain-of-function mutations in Aux/IAA-encoding genes, and both TPL and HDA19 are
recruited to activating ARF-binding sites specifically in low-auxin conditions (192, 224). TPL
recruits HDAC complexes in plants (102) (Figure 2), as has been reported for its metazoan counterparts
(24, 212). HDACs remove acetyl groups from lysines on histones (primarily histones H3
and H4), which leads to a more compact chromatin state and reduced accessibility of the genomic
DNA for transcription factors or the general transcriptional machinery (53). The compacted chromatin
prevents auxin-responsive gene expression when Aux/IAA levels are high and auxin levels
are low. An increase in auxin levels leads to Aux/IAA protein degradation and eviction of TPL
and HDAC from activating ARF target sites but leaves the compacted chromatin behind (224).
A recent study showed that another class of chromatin regulatory proteins, the SWITCH/
SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling ATPases, helps overcome
this repressed chromatin state upon auxin sensing (Figure 2). SWI/SNF chromatinremodeling
complexes use the energy derived from ATP hydrolysis to alter the occupancy or
positioning of nucleosomes on the DNA, thereby changing the accessibility of the genomic DNA
in the context of chromatin (33). The Q-rich middle region of the class A ARF5/MP physically
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SWI/SNF
HDAC
TPL
ARF
AUX/IAA
Deacetylation
DDDD
SWI/SNF
Ac Ac AcAcAc
HAT
DDDD
SWI/SNF
Eviction
a Low auxin concentration
b High auxin concentration
TFs
Histone
Figure 2
Control of gene expression by ARF proteins in a chromatin context. (a) Under low auxin concentrations,
Aux/IAA proteins complex with ARFs bound at target loci and recruit the TPL corepressor and histone
deacetylases (HDACs), resulting in a compact chromatin environment that prevents unlicensed auxin
response gene expression. Aux/IAA proteins also block the physical interaction between MP and the BRM
and SYD SWI/SNF chromatin-remodeling complexes. (b) Under high auxin concentrations, sensing of
auxin causes a rapid degradation of Aux/IAA proteins, which leads to loss of TPL and HDAC complexes and
allows SWI/SNF complexes to interact with ARFs (MP/ARF5). SWI/SNF-mediated chromatin remodeling
destabilizes nucleosomes near the MP-bound sites, freeing binding sites for additional transcription factors
(TFs). These factors bind to ARF target loci, which is followed by recruitment of histone acetyltransferases
(HATs). Protein abbreviations: ARF, AUXIN RESPONSE FACTOR; Aux/IAA, AUXIN/INDOLE-3ACETIC
ACID; BRM, BRAHMA; MP, MONOPTEROS; SWI/SNF, SWITCH/SUCROSE
NONFERMENTING; SYD, SPLAYED; TPL, TOPLESS. Additional abbreviation: Ac, acetylation.
interacts with and recruits plant SWI/SNF chromatin-remodeling complexes formed around
BRAHMA (BRM) or SPLAYED (SYD) to MP target loci (224). BRM and SYD are necessary
for auxin-responsive gene expression and execute this role by unlocking the repressed chromatin
state at MP target loci, which opens up binding sites for additional transcription factors. Under
low auxin, SWI/SNF remodeler recruitment is blocked by Aux/IAA proteins complexed with MP.
Intriguingly, tethering SWI/SNF complexes to MP target loci rescued morphological defects of
mp mutants, indicating that SWI/SNF complex recruitment is likely a major function of MP.
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Histone
acetyltransferases
(HATs): enzymes
that add acetyl groups
to lysines on histones,
promoting
transcriptional
activation
Additional chromatin regulators, such as histone acetyltransferases (HATs), likely act after this
step. Given that the chromatin-remodeling and histone (de)acetylation steps may take time to
execute, an interesting question is whether fast auxin responses—those occurring within minutes
(1)—are based on this mechanism.
QUANTITATIVE CONTROL OF TRANSCRIPTIONAL OUTPUT
IN RESPONSE TO AUXIN
As discussed above, the core auxin response machinery leads to both qualitatively and quantitatively
different responses in plant development. In this section, we summarize insights into how
interactions between core components and other factors determine the quantitative output of
auxin response.
Aux/IAA Degradation
Once auxin binds the pocket in the TIR1/AFB protein, the affinity for Aux/IAA proteins increases
(193). Aux/IAA proteins are subsequently ubiquitinated (118) and degraded in the 26S proteasome
(72, 234) (Figure 3). As the first step in Aux/IAA degradation, its binding by SCFTIR1/AFB
has received much attention; later steps have not been studied in as much detail. For example,
ubiquitination has been demonstrated in a protoplast assay (118), but the sites and extent of in
vivo ubiquitination are not well understood. Gilkerson et al. (68) recently showed that this step
may be more complex than initially thought. Even after mutating all 16 lysines in Arabidopsis
IAA1 and thus eliminating all possible canonical ubiquitin acceptor sites, they found that the protein
was still unstable and ubiquitinated. Drug treatments suggested that auxin-dependent IAA1
ubiquitination may involve noncanonical oxyester bonds to serines, threonines, or cysteines. As
each of these residues can be changed by posttranslational modifications (such as phosphorylation
or disulfide bridges), this work opens the exciting possibility that modifications to the Aux/IAA
protein can influence the competence of Aux/IAA proteins for auxin-dependent degradation.
Interestingly, the SCFTIR1/AFB
E3 ligase complex is itself also subject to dynamic, auxindependent
regulation. In a screen for mutations in TIR1 that change auxin-dependent interactions
with Aux/IAA proteins in yeast, Yu et al. (233) identified a mutation that increases TIR1-Aux/IAA
interactions. Closer inspection revealed that this mutation abrogated the incorporation of TIR1
in the SCF complex, and because TIR1 is itself ubiquitinated and degraded (188), the mutation
stabilized the protein (Figure 3). Because mutant TIR1 interacted with Aux/IAA proteins without
inducing their degradation, the mutation inhibited the auxin response in plants. This suggests
that, in the absence of auxin, autocatalytic turnover of TIR1 protein maintains the homeostasis of
SCFTIR1/AFB
complexes.
Until recently, evidence that such quantitative output, defined by the Aux/IAA degradation rate
(see The Players: Core Components That Translate Auxin Sensing to Transcriptional Responses,
above), determines biological output had been lacking. By generating versions of IAA14 with
different auxin-dependent degradation rates, Guseman et al. (76) tested the hypothesis that the
degradation rate determines the pace of progression through subsequent stages of lateral root
development. Previous studies had shown that this process depends on auxin response at several
steps (43, 64) and that the progression of lateral root development is defined by a set beginning and
several subsequent archetypical stages (117). After showing in a reconstituted yeast system that
engineered Aux/IAA proteins indeed have a range of degradation rates, Guseman et al. (76) assessed
the properties of the mutated proteins in plants. In transgenic plants, the Aux/IAA degradation
rate strongly correlated with the developmental progression of lateral root development. Thus,
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TIR1/
AFB1–5
ASK1
CUL1
RBX1
E2
U
Aux/IAA
N=O
miR393
Salt
flg22 Nitrate
Metals
Waterlogging
BIN2
P
miR847
Proteasome
AvrRpt2
ARF
Auxin
Figure 3
Regulation of auxin response output. Green arrows indicate positive regulation, and red lines indicate
inhibition. Auxin promotes interaction between the SCFTIR1/AFB complex ( gray) and Aux/IAA proteins
(red ), leading to the transfer of ubiquitin (U) (blue) to the latter and to degradation in the proteasome.
Aux/IAA proteins bind to and inhibit ARF transcription factors ( green), which can regulate transcription
when released from inhibition. The auxin response pathway involves feedback loops: ARFs activate
transcription of Aux/IAA proteins and trigger expression of microRNA 847 (miR847), which inhibits
Aux/IAA28 accumulation. In addition to ubiquitination, at least two other posttranslational modifications
alter auxin response: TIR1 can be S-nitrosylated (magenta), and the kinase BIN2 can phosphorylate (orange)
ARFs to inhibit their interaction with Aux/IAA proteins. Other signals also regulate auxin response: Levels
of TIR1 are controlled by autoubiquitination in the absence of substrate (dashed arrow) as well as by miR393,
which serves as an input hub for several environmental signals. Finally, the pathway can be hijacked by
Pseudomonas syringae, as its secreted effector protein AvrRpt2 promotes Aux/IAA protein degradation.
Protein abbreviations: ARF, AUXIN RESPONSE FACTOR; ASK1, ARABIDOPSIS SKP1
HOMOLOGUE; Aux/IAA, AUXIN/INDOLE-3-ACETIC ACID; BIN2, BRASSINOSTEROID
INSENSITIVE 2; CUL1, CULLIN 1; flg22, 22-amino-acid flagellin fragment; RBX1, RING-BOX 1;
TIR1/AFB, TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX.
plants seem to leverage the quantitative nature of the TIR1/AFB-auxin-Aux/IAA interaction to
generate quantitative auxin response output in development.
Transcriptional Regulation
Given the profound impact of auxin output on plant growth and development, it seems intuitive
that this output must be buffered and balanced to prevent excessive response (Figure 3). Indeed,
feedback control has been demonstrated at the level of auxin transport: PIN-FORMED (PIN)
auxin efflux carrier genes are transcriptionally upregulated by auxin such that, when cellular auxin
levels rise, excess auxin is transported out of the cell (208). A similar mechanism operates in auxin
biosynthesis regulation. The YUCCA (YUC) auxin biosynthesis enzyme genes are transcriptionally
repressed by auxin (191). Hence, high cellular auxin levels stall endogenous synthesis, and
lower auxin levels (as occur, for example, in yuc mutants or upon pharmacological inhibition of
YUC activity) lift transcriptional repression and elevate cellular auxin levels. Finally, Aux/IAA
genes were initially identified because they are transcriptionally upregulated by auxin treatment,
which suggested intrinsic feedback control (2). This feedback regulation has now been formally
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demonstrated using the MP/ARF5 protein: MP/ARF5 triggers activation of a subset of the 29
Aux/IAA genes through direct interaction with their gene promoters (101). The Aux/IAA proteins
encoded by these same genes directly interact with MP/ARF5 and inhibit its activity.
Posttranscriptional Regulation
In systematic studies identifying microRNAs in Arabidopsis following their initial discovery, a
microRNA specific to TIR1 and the AFB genes was also identified. This microRNA, miR393,
negatively regulates TIR1, AFB2, and AFB3 transcript and protein accumulation (130, 143). Under
normal growth conditions, miR393 expression acts to restrict TIR1/AFB protein levels (143),
and analysis of a miR393a miR393b double mutant, which has reduced levels of this microRNA,
demonstrated that this restriction of TIR1/AFB protein levels is required for normal auxin response
(219). Mutants showed a mildly increased response to auxin and subtle growth defects.
Because some miR393 remains even in the double mutant, it is difficult to evaluate the extent of
microRNA regulation and its impact on auxin signaling, but it is clear that this regulatory node can
act as a nexus for environmental control of auxin response (Figure 3). The miR393 genes are controlled
by a variety of environmental conditions, including salt stress (89), waterlogging in maize
(113), metal toxicity (121), nitrate (207), and bacterial flagellin (130). Thus, miR393-TIR/AFB
may represent a module for environmental control of auxin responses. In the case of flagellin
regulation, Navarro et al. (130) showed that the increased miR393 expression and concomitant
decrease in auxin response restricted growth of the bacterial infection, and it is likely that other
stimuli likewise tailor growth through a primary effect on auxin response.
The impact of small-RNA regulation on auxin response is not limited to the TIR1/AFB receptors.
In fact, several small RNAs target ARF transcripts. miR160 targets ARF10, -16, and -17 (210),
whereas miR167 antagonizes ARF6 and -8 (109). Finally, miR390 phases trans-acting small interfering
RNAs from the TAS3 locus that in turn target ARF2, -3, and -4 (3), which may contribute
to auxin-controlled accumulation of ARF2, -3, and -4 messages (30, 119, 230). For each of these
small RNAs, it has been well established that regulation impacts the activity of the respective ARFs
during development (reviewed in 147). Wang & Guo (209) recently found the first microRNA
targeting an Aux/IAA protein: miR847, which is transcriptionally upregulated by auxin in a TIR1dependent
manner and targets IAA28 mRNA for cleavage (Figure 3). Thus, auxin clears cells not
only of IAA28 protein (166) but also of its mRNA, thereby prolonging the duration of the auxin
response in leaf formation. This regulation is the opposite of that performed by all other small
RNAs that target components of the auxin response, as those all antagonize positive regulators
and thereby temporally restrict auxin response. It appears that a small RNA regulatory layer acts
to sculpt the auxin response and integrate it with environmental cues.
Posttranslational Control
Since the discovery of the main components in nuclear auxin signaling, various studies have explored
regulation by posttranslational control, including in vitro S-nitrosylation of TIR1 (194),
in vitro phosphorylation of Aux/IAA proteins by phytochrome (36), and potential cis-trans isomerization
of prolines in Aux/IAA proteins (47). However, none of these modification have been
demonstrated to occur in vivo, and their impact on auxin responses remains to be determined. A
phosphorylation event in ARF7 and ARF19 does appear to be important for their function in lateral
root development (31): In a semi–in vivo assay, both ARF proteins are directly phosphorylated
by the GLYCOGEN SYNTHASE KINASE (GSK) kinase BRASSINOSTEROID INSENSITIVE
2 (BIN2), which acts in brassinosteroid signaling (Figure 3). Phosphorylation inhibits
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Aux/IAA-ARF interactions and potentiates ARF activity. Surprisingly, in this context BIN2 is
activated not by brassinosteroids but by the peptide TRACHEARY ELEMENT DIFFERENTIATION
INHIBITORY FACTOR (TDIF). Intersignal crosstalk is widespread in plant signaling
(45), yet this is this first example of a signal that acts through posttranslational modification of an
ARF protein. An interesting question for future studies is whether this is a more general mode of
regulation in the auxin signaling pathway.
Finally, pathogens have also found a way to tap into the auxin response machinery. Pseudomonas
syringae injects pathogen effector proteins into infected host cells; among these injected
proteins is AvrRpt2, which interferes with auxin responses (27). A detailed study showed that
AvrRpt2 promotes Aux/IAA protein degradation and that this degradation-inducing activity acts
independently of auxin-promoted degradation (40). Intriguingly, a mutation in IAA7 that prevents
auxin-dependent degradation (axr2-1) reduces AvrRpt2 virulence, suggesting that Aux/IAA
degradation is necessary for successful pathogen colonization and highlighting a successful strategy
of hijacking the auxin response pathway. Similarly, the Tobacco mosaic virus replicase protein
interacts with and inhibits Arabidopsis host IAA26 protein, and the resulting reprogramming of
auxin response contributes to viral pathogenicity (142).
Protein Interactions and Oligomerization
Because the TIR1/AFB, Aux/IAA, and ARF families are all subject to transcriptional and posttranslational
regulation, their relative levels in cells differ. If proteins have diversified in their
affinity toward auxin and/or other proteins in the pathway, then interactions among components
could potentially generate different responses. Interactions among components have been experimentally
tested, and the results do indeed support diversification, at least in a quantitative sense.
The intrinsic preference of TIR1/AFB members for auxin (21) and Aux/IAA substrates as well as
differences in the degradation rates of the latter (82) suggest that different concentrations of auxin
in the same cell, or the same concentrations of auxin in different cells, result in specific cellular
concentrations of several Aux/IAA proteins (Figure 4). How are these differences translated to
gene regulation? Aux/IAAs act by binding to and inhibiting ARF transcription factors (195). An
important question is how specific levels of Aux/IAA proteins affect the ARF factors present in a
cell. Systematic efforts have shown that almost all Aux/IAA proteins can interact with a subset of
ARFs with a long, Q-rich middle region (i.e., activating ARFs), whereas interactions with other
ARFs are limited and more specific (206). A recent study that incorporated coexpression data
of ARF and Aux/IAA genes came to a similar conclusion (146). The emerging picture is a simple
one: The totality of the accumulating Aux/IAA proteins converges on the Q-rich ARF proteins
and collectively defines the degree of activity (Figure 4). However, the Aux/IAA-ARF assays that
have been reported so far (146, 206) were not quantitative, and it is possible that the interaction
affinities differ by orders of magnitude. Another important point is that most of the interactions (or
lack thereof ) have not been tested in plant cells, which misses the contribution of posttranslational
modifications to the physical interactions. Significant differences in affinity could allow auxin to
activate different ARF proteins at different concentrations. A quantitative relationship between
auxin dose and gene activity has not yet been established, and whether there are separable gene
sets that respond only to low or high auxin concentrations remains to be seen.
Both Aux/IAA and ARF proteins oligomerize in vitro, both in crystals and in solution. Mutations
that prevent oligomerization affect the ability of Aux/IAA16 to inhibit auxin response
(100), and analogous mutations in ARF5 make the protein less sensitive to auxin inhibition (129).
Whether the biological activity of Aux/IAAs and ARFs requires oligomerization is an interesting
open question, but proteins do have the potential to oligomerize. Intriguingly, structural studies
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TIR1/
AFB1–5
ASK1
CUL1
RBX1
E2
Aux/IAA
1
2
3
4 ARF
Auxin
Cofactors
Figure 4
Specificity in auxin response. Transcriptional auxin output depends on interactions and regulation at various
levels, ultimately leading to either quantitatively or qualitatively different gene expression profiles. ( ) The
affinity of the TIR1/AFB-auxin-Aux/IAA interaction depends on the identity of the receptor, the type of
auxin molecule, and the identity of the Aux/IAA protein and can thus vary by orders of magnitude.
( ) Aux/IAA-ARF interactions through their homologous C-terminal domains are likely selective.
Aux/IAAs preferentially interact with class A ARFs, although interactions with class B and C ARFs have also
been demonstrated. The affinities among the families likely depend on the exact pairs. ( ) The selection of
DNA target sites by ARF-DNA interactions can be selective not only by direct recognition of binding sites,
but also by the spacing between two adjacent inverted binding sites to which ARF dimers can bind with high
affinity. Although ARFs bind nearly identical motifs in vitro, there may be more selectivity in vivo. The
optimal spacing between binding sites differs, at least in vitro, between ARFs, which adds selectivity.
Furthermore, ARFs may theoretically heterodimerize, further expanding the range of binding specificities.
( ) ARF-interacting cofactors can alter ARF activity or DNA-binding specificity. Protein abbreviations:
ARF, AUXIN RESPONSE FACTOR; ASK1, ARABIDOPSIS SKP1 HOMOLOGUE; Aux/IAA, AUXIN/
INDOLE-3-ACETIC ACID; CUL1, CULLIN 1; RBX1, RING-BOX 1; TIR1/AFB, TRANSPORT
INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX.
of the TPL tetramer revealed that it preferentially complexes with oligomerized EAR motif–
containing interaction partners (96); thus, Aux/IAA oligomerization may result in enhanced TPL
and HDAC recruitment. Based on these observations, Farcot et al. (57) developed a model of the
auxin response pathway composed of ordinary differential equations that describe interactions between
TIR1/AFB, auxin, Aux/IAAs, and ARFs. Mathematical analysis of this network suggested
that Aux/IAA-ARF interactions determine the response amplitude, whereas Aux/IAA-Aux/IAA
interactions set the speed of the response and ARF-ARF interactions determine the sensitivity.
Thus, oligomerization could significantly affect the output of auxin response, as all output parameters
(amplitude, speed, and sensitivity) depend on interactions mediated by the domain that can
oligomerize.
GENERATING SPECIFICITY IN AUXIN RESPONSES
Auxin triggers various distinct responses in plant development. Auxin accumulation in the shoot
apex triggers floral organogenesis (85, 157, 206), in root pericycle cells induces lateral root formation
(22), and in embryos induces either cotyledon or root initiation depending on whether the
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accumulation is at the top or bottom of the embryo (123). Thus, the response machinery must
accommodate such local interpretation of auxin accumulation, and a key question is how hormone
perception is locally translated to gene expression. Functional specificity can be generated
at several levels (Figure 4), each of which we discuss below.
Protein-DNA Interactions Select Auxin-Responsive Genes
Auxin responses are functions of the genes that are activated or repressed in response to auxin.
Thus, DNA recognition by ARF transcription factors is an important step in selecting the auxinresponsive
gene repertoire. ARFs were originally identified based on their ability to bind a
model AuxRE (TGTCTC) (199, 202) that was identified through promoter analysis of an auxinresponsive
gene in soybean (114). Interestingly, following the identification of an AuxRE in the
mid-1990s (202) and the subsequent demonstration that ARFs bind this sequence (199, 201),
there had not been an exhaustive screen for DNA sequences bound by ARFs until recently.
Clearly, the presence of the canonical TGTCTC AuxRE in promoters is insufficient to explain
auxin-responsive transcription in vivo (97, 122), and unbiased bioinformatics analysis suggests
that more complex motifs mediate auxin responsiveness (122). These analyses also suggest that
auxin-regulated gene activation and gene repression are mediated by distinct motifs.
The first efforts to define the full range of sequences bound by ARFs were recently published.
Both made use of protein-binding microarrays and a recombinant ARF protein (17, 61). Interestingly,
in both studies, the optimal ARF binding site was distinct from the TGTCTC site identified
15 years earlier (201). In fact, Boer et al. (17) suggested that TGTCTC represents a mediumaffinity
target site and that the novel motif TGTCGG is a high-affinity binding site. Indeed, when
Liao et al. (107) used the TGTCGG site to generate a gene expression reporter analogous to
the DR5 reporter based on TGTCTC, the high-affinity element showed broader activity in vivo,
revealing suspected sites of auxin response, which could be explained by its approximately tenfold
higher sensitivity to auxin.
Structural studies of the ARF DNA-binding domain suggested an additional level of control
in selecting auxin-dependent genes (17). Although selection of the DNA element by the ARF
DNA-binding domain is dictated by the DNA-contacting residues in the protein-DNA interface,
DNA binding is defined by more than just these interactions. Because DNA-binding domains homodimerize,
ARF dimers can bind complex motifs that have two binding sites in an inverted constellation
with defined spacing (Figure 4). Ulmasov et al. (199) demonstrated almost two decades
ago that such motifs are highly efficient in mediating auxin-dependent transcription. Structural
biology has now suggested a basis for this efficiency, and Boer et al. (17) indeed showed that ARFs
bind such complex sites cooperatively. High-affinity binding requires both two adjacent binding
sites and an intact ARF dimerization interface (17), which suggests that ARFs can bind either as
monomers to single motifs with low affinity or as dimers to complex motifs with high affinity.
A remaining question is how differences between ARF proteins help the selection of distinct
sets of target genes, and in this sense, it was surprising that all ARFs tested with a protein-binding
microarray showed nearly identical binding sites (17, 61). However, when tested for binding
affinity in complex motifs, ARF1 and ARF5 dimers bound more or less strongly depending on
the length of the spacer between the two inverted binding sites: ARF1 could bind only when the
spacing was 7 or 8 based, whereas ARF5 bound a longer range of spacing (17). Thus, spacing
may help discriminate ARF binding. This work has also raised many new questions: Do ARFs
heterodimerize via the DNA-binding domain? Do ARFs differ in their in vivo binding sites? Does
spacing between binding sites discriminate ARFs in vivo? Clearly, the answers to these questions
will help researchers understand how ARFs select genes for local responses to auxin.
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Binding site selection can be influenced by the affinity of the transcription factor for a given
site, the number of sites present, and their accessibility in the context of chromatin. Recent studies
in Drosophila have uncovered an inverse relationship between the affinity of the binding site and
its specificity for a given transcription factor (that is, low-affinity binding sites have the highest
transcription factor specificity); moreover, clustered low-affinity sites contribute to the robustness
of the response (39). Indeed, several reports have suggested that the AuxRE core (TGTC), which
may represent a low-affinity binding site, is sufficient for ARF recruitment (127, 228). The evolutionarily
conserved AuxRE core elements in the regulatory region of LEAFY (LFY ) are bound
only by MP during reproductive development, not in seedlings, even though MP binds to other
target genes at this stage (228). One possible explanation for this finding is that sites in LFY are
inaccessible during the vegetative phase; alternatively, a higher MP level may be necessary to
bind these low-affinity sites (228). In agreement with the latter idea, MP levels increase during
development (228).
Importantly, the ARF family has the potential to accommodate distinct outputs: The gene
expression of the family is developmentally regulated, with cells expressing different combinations
of ARFs (153). Furthermore, several ARFs mutate to create distinct loss-of-function
phenotypes despite coexpression of (close) homologs (80, 153). Finally, promoter-swap experiments
have shown that there is a limited potential for one ARF to replace a second, mutated
ARF (152, 153, 214). Thus, auxin accumulation is locally translated to gene expression
changes through an interaction network that culminates in functionally distinct ARF transcription
factors.
Cofactors Can Modulate Auxin Output
Transcription factors often act in protein complexes, where cofactors can alter the activity or
DNA-binding specificity of DNA-binding transcription factors (Figure 4). In some cases, cofactor
binding can define novel specificities that enable the DNA-binding factors to bind DNA that it
normally would not (184). Likewise, ARF-interacting proteins could conceivably modulate activity
or specificity. Surprisingly few interactions with ARFs have been reported. MYB77 interacts with
ARF7 and is required for the auxin-dependent activation of several auxin-dependent genes (182).
It is not clear how general this role of MYB77 in auxin signaling is, but mutant phenotypes
suggest that it may be restricted to lateral root formation (182). Furthermore, MYB77 appears to
connect abscisic acid signaling to auxin responses (236), which may be an example of modulation
of a specific local auxin response. The basic helix-loop-helix (bHLH) factor BIG PETAL (BPE)
interacts with ARF8, and the genes encoding these two proteins both regulate petal growth (205).
How BPE modulates ARF function remains unclear.
Oh et al. (134) recently showed that ARF6 interacts with the bHLH protein PHYTOCHROME
INTERACTING FACTOR 4 (PIF4) and the transcription factor BRASSINAZOLE
RESISTANT 1 (BZR1). Chromatin immunoprecipitation sequencing (ChIP-seq) analysis of these
three proteins showed a considerable overlap in genomic target sites, and auxin regulation of a
large number of genes (presumably by ARF6) depends on PIF4 and BZR1 function. This work
shows the interdependence of these proteins and suggests that ARF6 function may require the
assembly of a transcription complex, at least in the context of hypocotyl growth. Again, it is unclear
how PIF4 and BZR1 influence ARF6 activity or specificity, but the complex presents a good model
to address mechanistic aspects of cofactor function. Strikingly, all three examples mentioned here
involve closely related members of the ARF proteins with a Q-rich middle region, and an interesting
question is whether interactions with other transcription factors are limited to this clade of
ARFs.
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Chromatin-Level Control in Auxin Responses
Chromatin-level regulation may also provide a means to generate specificity in auxin responses.
The interactions between auxin-dependent genes, response components, and chromatin have not
been explored in detail, but there are several indications that such interactions occur. A new mechanism
for modulation of auxin output was suggested by the recent discovery that the activating
ARF MP unlocks chromatin at its targets together with SWI/SNF chromatin remodelers (224). In
this study, Wu et al. (224) showed that swi/snf mutants phenocopy mp mutant phenotypes and that
their activity is required for MP to activate its target genes. Chromatin unlocking allows transcriptional
regulators to contact cognate binding sites that were previously occluded (Figure 2). These
secondary transcriptional regulators can contribute to the specificity of the auxin response if their
accumulation and activity are cell type or condition specific (224). One corollary of this finding is
that evolutionarily conserved cis-regulatory elements should be present next to AuxREs or MPbound
sites. Although this has not been exhaustively tested, such co-occurrence has been reported
(13, 224). The sequence-specific binding proteins, whose access to DNA is auxin gated, may also
recruit additional chromatin regulators. Histone acetylation and transcriptional activation of MP
targets is dependent on prior chromatin unlocking (224). Intriguingly, at a target locus that was
tested in detail, an evolutionarily conserved basic leucine zipper (bZIP) motif was occluded by
the chromatin prior to unlocking (224). In another study, the bZIP11 transcription factor, which
binds near an AuxRE in the GH3 promoter and helps activate the GH3 gene, recruits a HAT to
the promoter to bring about gene activation (216).
In addition, several PLANT HOMEODOMAIN (PHD) finger proteins—OBERON 1
(OBE1) and OBE2 and TITANIA 1 (TIT1) and TIT2—are required for normal development,
and mutants show defects similar to mp/arf5 and bodenlos (bdl )/iaa12 mutants (170, 171). Although
MP/ARF5 is still normally expressed in obe and tit mutants, several MP/ARF5 target genes are
downregulated in a region-specific manner. OBE1 binds to the promoter of the MP/ARF5 target
gene TARGET OF MONOPTEROS 7 (TMO7) in a region that overlaps with the MP/ARF5 binding
site (177). Hence, OBE and TIT proteins are required for the activation of ARF target genes.
PHD fingers are known to bind methylated lysines on histone proteins (105), and it is thus likely
that the histone modification status at target loci is integrated into auxin-dependent regulation.
It will be interesting to assess what link (if any) exists between these histone-binding proteins and
chromatin remodeling or histone modification.
TRANSCRIPTIONAL CONTROL OF AUXIN-DEPENDENT PROCESSES
Inspired by phenotypes in auxin response mutants (204), the identity of genetically defined developmental
regulators as auxin response components (80), or the expression of auxin response
reporter genes (62, 168), researchers have studied the involvement of auxin in several developmental
processes. In this section, we focus on a few of these processes in which auxin action has been
linked to individual and causal target genes. We discuss qualitative aspects of auxin-controlled
development here. For dose-dependent outputs, see the sidebar Quantitative Auxin Output: A
Plant Morphogen?
Embryogenesis
Life in flowering plants arguably begins at fertilization. Subsequent steps turn a zygote into a
mature embryo that includes precursors for the major tissues as well as root and shoot meristems
to sustain postembryonic growth (213). It has long been recognized that several steps in embryogenesis
depend on normal auxin activity (Figure 5). Treatments of in vitro–grown embryos
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QUANTITATIVE AUXIN OUTPUT: A PLANT MORPHOGEN?
The auxin response machinery has the potential to generate different responses to different auxin concentrations:
TIR1/AFB-Aux/IAA interactions have affinity constants that depend on both partners (21), and the binding equilibrium
therefore varies with the auxin concentration in different ways for each Aux/IAA protein. In addition, the
degradation rates of all Aux/IAA proteins are not the same (52, 82, 125), which means that after an auxin pulse,
the differential clearance of Aux/IAA proteins in a cell can create a dynamically changing mix of Aux/IAA proteins
over time. Finally, Aux/IAA-ARF interactions have some (perhaps limited) selectivity (146, 206, 214), and ARFs are
biochemically distinct (152, 214) and expressed in different patterns (153). Thus, dynamic Aux/IAA landscapes can
translate into different ARFs being activated to varying degrees.
Gradients of signaling molecules are known to control development in various animals, with famous examples
being Decapentaplegic (Dpp) (161), Bicoid (148), and Sonic Hedgehog (Shh) (34). These molecules, collectively
termed morphogens, locally induce responses along the gradient and thereby enable cells to “read” the local
concentration. Auxin gradients have been widely discussed (204), but such gradients often do not extend over long
ranges; instead, they are defined by a few cells with high DR5 reporter activity and rapidly declining or absent
reporter activity in neighboring cells (62, 85, 168). These are technically gradients, but the question that remains
unanswered is whether distinct outputs are triggered by different auxin concentrations along such a gradient.
No experiments have been reported that directly address the questions of whether different auxin concentrations
trigger expression of unique gene sets and whether cells could therefore interpret their position in a gradient of
auxin. Particularly with the advent of cell type–specific profiling of auxin responses (7), answers to these questions
should soon be within reach.
either with auxin or with auxin transport inhibitors cause distinctive defects in different species,
including problems in the formation of the apical-basal axis, the formation of the root or shoot,
and the formation or separation of cotyledons (77, 108). Indeed, both initial and improved gene
expression reporters for auxin activity are active during several steps of embryogenesis and in
different sites (62, 107) (Figure 5). Genetic analysis of auxin biology has revealed that all aspects
of auxin action—synthesis, influx, efflux, and response—are critical for normal embryogenesis.
Higher-order mutants in auxin biosynthesis genes cause a distinctive rootless defect (29, 187), and
very similar defects occur in higher-order mutants in auxin influx carriers (164) and components
of the SCFTIR1/AFB
complex (48, 49, 87). Mutations in PIN auxin efflux regulators and in the PIN
polarity regulator GNOM lead to overlapping but distinct phenotypes (220), consistent with the
expected and observed dual effect of transport inhibition: Auxin does not sufficiently accumulate
in target cells yet accumulates excessively in source cells.
Several defects induced by reduced auxin activity can be explained by altered activity of only a
few of the 29 Aux/IAAs and 23 ARFs. A loss-of-function mutation in MP/ARF5 causes an absence
of the embryonic root and hypocotyl as well as of much of the vascular tissue (14). An mp/arf5
arf7 double mutant also eliminates cotyledon formation (81). Likewise, a stabilizing mutation in
the BDL/IAA12 protein that prevents its auxin-dependent degradation (49, 78) causes a nearly
identical spectrum of phenotypes (79), which suggests that the interacting MP/ARF5-BDL/IAA12
protein pair (215) is a major effector of auxin action in the embryo.
Beyond MP/ARF5, the Arabidopsis genome encodes 22 other ARF proteins, and a systematic
analysis of transcription patterns revealed that several of these are expressed in the embryo (153).
In fact, ARF gene expression patterns are diverse and suggest that most cell types express unique
sets of ARF transcription factors. Given that ARF proteins are not simply interchangeable (see
Generating Specificity in Auxin Responses, above), this finding suggests that auxin accumulation
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Shoot apical
meristem,
cotyledons
Hypocotyl, root
Root cap
Low High
DR5v2 activity
Octant Early globular Transition
ARF(s)
Target(s)
and
pathway
Process
?
?
Cell
division
orientation
ARF5 ARF9/? ARF5 (and -7?)
TMO5
LOG4
CK
NTT
WIP4
WIP5
TMO7
? DRN WRKY23
Vascular
cell
division
Hypophysis/
root
formation
Suspensor
identity
Cotyledon
formation/
separation
Cotyledon/
root
formation
Figure 5
Auxin response during embryogenesis. The sites of auxin response vary at different stages of embryogenesis
(shown chronologically from left to right), as shown by expression of the DR5v2 reporter (107). These sites
coincide with the establishment of regions destined to become organs in the seedling (thin lines between stages
demarcate region boundaries). The table at the bottom summarizes the ARF or ARFs that act at each stage
to control target genes and pathways, ultimately directing cellular or developmental processes. Protein
abbreviations: ARF, AUXIN RESPONSE FACTOR; CK, cytokinin; DRN, DORNR ¨OSSCHEN; LOG4,
LONELY GUY 4; NTT, NO TRANSMITTING TRACT; TMO, TARGET OF MONOPTEROS;
WIP, WIP DOMAIN PROTEIN.
in different parts of the embryo triggers different transcriptional responses. To determine whether
there are indeed multiple local auxin responses, Rademacher et al. (152) expressed the bdl/iaa12
mutant protein from a range of tissue-specific promoters in the embryo. Because Aux/IAA-ARF
interactions have limited specificity (146, 206, 214), this strategy allowed the authors to generically
inhibit ARFs expressed in the target cell. The results confirmed all known auxin-dependent
processes and revealed several novel ones (152). These include the prevention of embryo formation
in suspensor cells and control of cell division orientation in the eight-cell embryo (231). An
important question now is which target genes mediate local responses to auxin.
Embryogenesis is a fundamentally important phase of plant life, yet very few auxin-dependent
genes have been described in this process, likely because of the embryo’s small size and lack
of accessibility. However, studies of postembryonic functions of MP/ARF5 have isolated several
direct target genes, some of which are functionally important for auxin-dependent embryogenesis.
In an effort to isolate genes that are controlled by both MP/ARF5 and BDL/IAA12, Schlereth
et al. (177) identified a set of likely direct transcriptional targets (Figure 5); these targets included a
large number of uncharacterized factors, and their detailed study of four of these factors (TMO3,
-5, -6, and -7) showed that some indeed contribute to MP/ARF5-dependent embryonic root
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formation. Expression of all four genes is strongly downregulated in mp/arf5 mutant embryos; at
least the TMO3, -5, and -7 promoters are bound by MP/ARF5 in vivo; and restoring expression
of TMO5 or TMO7 partially rescued the phenotype of a mutant with a weak allele of mp/arf5.
Downregulation of TMO7 caused mp/arf5-like embryonic root defects. Finally, TMO7 appears
to be transported from its domain of expression to neighboring cells to induce the formation of
the future quiescent center (177).
OthertranscriptionfactorshavebeenlinkedtoMP/ARF5functioninamoredirectedapproach.
Both DORNR ¨OSSCHEN (DRN) and WRKY23 are expressed in the embryo in a subdomain of
the MP/ARF5 domain (35, 74), and because DRN gene expression is auxin dependent (35) and
WRKY23 loss-of-function phenotypes resemble those of the mp/arf5 mutant (74), Cole et al. (35)
and Grunewald et al. (74) analyzed their regulation by MP/ARF5. In both cases, expression was
downregulated in mp/arf5 mutant embryos, and in one case (DRN), MP/ARF5 bound the target
promoter. Thus, a handful of direct MP/ARF5 targets have been identified, but there remains a
large gap in our understanding of how auxin triggers embryo development through MP/ARF5.
An intriguing facet of MP/ARF5 action in embryogenesis is that it appears to act non-cellautonomously.
MP/ARF5 is strongly expressed in cells in the lower half of the embryo, adjacent
to the hypophysis cell, which later forms the quiescent center (80, 215). Yet cell division
defects in mp/arf5 mutants occur in both the proembryo and the hypophysis (14). Two parsimonious
explanations could account for this discrepancy: (a) MP/ARF5 is expressed at very low
(nearly undetectable) levels in the hypophysis and triggers hypophysis specification locally, or (b)
MP/ARF5 triggers a nonautonomous signal that moves to the adjacent hypophysis. A recent paper
favored the former hypothesis (37). The NO TRANSMITTING TRACT (NTT ), WIP DOMAIN
PROTEIN 4 (WIP4), and WIP5 genes (collectively termed NWW) act redundantly in embryonic
root formation; an nww triple mutant is rootless (37). All three genes are expressed in the hypophysis
and its descendants. Expression is lost in the mp/arf5 mutant, and ChIP suggests that the genes
may be direct MP/ARF5 targets. Both in situ hybridization and a functional fluorescent protein
fusion to MP/ARF5 reveal low-level expression in the hypophysis, and a model has been proposed
in which MP locally activates NWW genes to control root initiation (37). Reality is probably more
complex, though; Weijers et al. (215) had previously shown that inhibition of MP/ARF5 in the
proembryo alone is sufficient to cause the mutant hypophysis defect, and complementation of the
mutant by expression of MP/ARF5 only in the proembryo domain completely rescued the mp/arf5
mutant defect. Thus, although MP/ARF5 may be expressed at low levels in the hypophysis (37), it
is clearly not required in this cell (215). The identification of more direct target genes should help
resolve whether MP/ARF5 acts directly on hypophysis formation or through cell-cell signaling.
Vasculature
Auxin has long been known to promote vascular tissue formation and differentiation. Classical
experiments by Sachs (169) showed that new vascular strands are formed from a local external
sourceofauxinappliedtoastemsegmentandthatthesestrandseventuallyconnecttothemainstem
vascular system. Thus, an auxin source is sufficient to direct vascular tissue formation. Likewise,
veins in leaves form along paths of increased auxin concentration defined by PIN-dependent
transport (175). In another classical model for vein formation, during graft union establishment,
cells express the DR5-GFP auxin response reporter well before new vascular tissue is formed
(120). Genetic evidence has shown that an auxin response is indeed also required for vascular
tissue formation: The mp/arf5 mutant makes very little vascular tissue (151).
One key question is how auxin, through MP/ARF5, triggers vascular tissue formation. Some of
its functions may be mediated by activation of PIN1 transcription, thus promoting vein continuity
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(178, 217). However, MP/ARF5 activity must also somehow trigger a gene expression program that
defines vascular tissue. Several direct MP/ARF5 target genes are specifically expressed in vascular
tissue and may mediate auxin-dependent vascular tissue formation. ARABIDOPSIS THALIANA
HOMEOBOX 8 (ATHB8) is directly activated by MP/ARF5 and is expressed early in procambial
cells throughout the plant. A mutation in ATHB8 affects normal leaf vein formation but is certainly
not critical for the process. Rather, vascular domains are wider in the mutant, which suggests that it
may restrict vascular tissue formation to define narrow veins (51). Similarly, Dof5.8 is activated by
MP/ARF5 and expressed in procambial cells (98). A mutation in Dof5.8 enhances a weak mp/arf5
allele, which suggests a functional contribution to leaf vascular tissue formation. Interestingly,
overexpression of the transcriptional repressor Dof5.8 reduces vascular tissue complexity (99),
which means that this factor is not an important MP/ARF5 output in vascular tissue formation.
Finally, among the genes isolated as MP/ARF5 targets (177) were two closely related bHLH
genes—TMO5 and TMO5-LIKE 1 (T5L1)—that showed vascular-specific gene expression (42).
Mutant analysis showed that TMO5, T5L1, and their close homologs T5L2 and T5L3 are collectively
required for the promotion of oriented, periclinal divisions in the vascular tissue of the
embryo and root (42). Such periclinal divisions are necessary to generate a vascular bundle from
the four precursor cells in the early embryo, and importantly, these divisions are strongly reduced
in the mp/arf5 mutant (42). TMO5 and its paralogs act as heterodimers with the bHLH
factor LONESOME HIGHWAY (LHW) (42, 136). The overlap of the auxin-dependent TMO5
and auxin-independent LHW transcription patterns defines a small zone in which periclinal divisions
occur (42), and joint overexpression of TMO5 and LHW can induce periclinal divisions
ubiquitously (42, 137). Recently, two studies showed that TMO5 and LHW promote periclinal
divisions by activating local cytokinin biosynthesis through the LONELY GUY 4 (LOG4) gene
(41, 137). Thus, an important output of MP/ARF5 activity in vascular cells is the activation of
periclinal division through its target TMO5 and subsequent cytokinin biosynthesis. Nonetheless,
the auxin-dependent vascular genes so far have not shed light on how auxin can trigger vascular
tissue identity, which remains a future challenge.
Organogenesis from the Reproductive Shoot Apex
A classical auxin response is organogenesis from the reproductive shoot apex. At the shoot apex, the
central stem cell pool gives rise to stem cell descendants at its flanks (8, 213). These cells become
competent to give rise to new lateral organs (leaves or flowers) when they perceive an auxin
maximum (157) (Figure 6). The most dramatic mutant phenotypes have been observed in flower
primordium initiation. Mutants in auxin biosynthesis, transport, and response all form pin-like
inflorescences that lack flowers (28, 138, 151, 206). These auxin maxima, which presage the sites
of future primordium initiation, are generated by the combined activities of the auxin efflux carrier
PIN1, the auxin influx carrier AUX1, and the PINOID kinase, which regulates PIN1 localization
(11, 32, 63, 138, 158). The relative positions of the newly initiated primordia (the phyllotaxis) are
species specific, and modeling, cell biological, and genetic studies have shown that they rely on
both a local auxin maximum and a region of low auxin concentration around it (6, 93, 185). The
only ARF linked to flower primordium initiation thus far is the activating MP/ARF5 (151).
Auxin likely is not solely responsible for selecting the site of organogenesis. Two cytokininupregulated
genes, encoding the response regulators ARABIDOPSIS RESPONSE REGULATOR
7 (ARR7) and ARR15, were identified as MP target genes in the shoot apex (237) (Figure 6).
MP bound to the promoter of ARR15 in vivo, and auxin and N-1-naphthylphthalamic acid (NPA)
treatment caused decreased and increased expression of ARR7 and ARR15, respectively, suggesting
that both genes are repressed upon auxin sensing. Accordingly, mutations in the AuxREs of the
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PIN1-GFPPIN1-GFP
MPAHP6
ARR7/15 TMO3/CRF2
LFY
PLT/ANT/AIL6 FIL
Cytokinin
response
Floral
identity
Cell division
and growth
Organ
polarity
Movement
Flower primordium initiation
Figure 6
MP-dependent flower primordium initiation. In the Arabidopsis inflorescence (top left), flowers arise in a
regular phyllotactic pattern from stem cell descendants at the shoot apex. The position of each primordium
is determined by a local maximum of the hormone auxin, marked by PIN1-GFP ( green fluorescence in bottom
left panel; image reproduced from Reference 228). In primordia, auxin activates MP, which induces the
expression of several direct targets whose activities converge on flower primordium initiation. AHP6, an
inhibitor of cytokinin signaling, is activated by MP and moves outside of the primordium zone to restrict the
cytokinin response to the primordium. MP also represses the expression of two negative regulators of
cytokinin response, ARR7 and ARR15, in the central region of the shoot apex and induces expression of an
AP2 family transcription factor gene, TMO3/CRF2, that has been linked to the promotion of cytokinin
responses in incipient flower primordia. MP promotes cell division and growth through direct activation of
two members of the PLT family of transcription factor genes, ANT and AIL6. MP induces expression of the
helix-turn-helix transcription factor gene LFY, which directs establishment of floral identity. Finally, MP
activates expression of the YABBY transcription factor gene FIL, which plays a role in specifying abaxial or
peripheral fate. Protein abbreviations: AHP6, ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER 6;
AIL6, AINTEGUMENTA-LIKE 6; ANT, AINTEGUMENTA; AP2, APETALA 2; ARR,
ARABIDOPSIS RESPONSE REGULATOR; CRF2, CYTOKININ RESPONSE FACTOR 2; FIL,
FILAMENTOUS FLOWER; GFP, green fluorescent protein; LFY, LEAFY; MP, MONOPTEROS;
PIN1, PIN-FORMED 1; PLT, PLETHORA; TMO3, TARGET OF MONOPTEROS 3.
ARR15 promoter caused increased expression. Finally, knockdown of these two negative regulators
of cytokinin response partially rescued organ initiation in mp null mutants (198). These and
additional data led to the hypothesis that cytokinin and auxin act in concert during organogenesis
in the shoot (9, 218, 232). Further support for this idea comes from more recent studies demonstrating
that MP directly activates the ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER 6
(AHP6) cytokinin signaling inhibitor, which non-cell-autonomously lowers the cytokinin response
in the region surrounding the auxin maximum (Figure 6). Loss of AHP6 caused the initiation of
multiple flowers from a single auxin maximum, suggesting that a reduction of cytokinin response
contributes to the generation of an inhibitory field that restricts the potential for organogenesis
(15). These data are consistent with the idea that reduced cytokinin signaling and reduced auxin
signaling in the zone surrounding an initiating primordium are required for the stereotypic spatial
initiation of flower primordia.
How is the auxin cue transmitted from auxin-activated MP to direct the cell identity changes
that culminate in the formation of a flower primordium? This reprogramming requires establishing
the primordium developmental program and erasing the meristem program. Expression of
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key regulators of plant development changes dramatically during the early stages of flower initiation;
for example, expression of the polarity gene FILAMENTOUS FLOWER (FIL) and the floral
identity gene LFY increases, whereas that of the organ boundary gene CUP-SHAPED COTYLEDON
2 (CUC2) and the meristem regulator SHOOT MERISTEMLESS (STM) decreases (85).
Genetic analyses had not identified factors acting directly downstream of MP in this process. The
first insight into why this was the case came from identification of the genes encoding the LFY
transcription factor and the PLETHORA (PLT) family transcription factors AINTEGUMENTA
(ANT ) and AINTEGUMENTA-LIKE 6/PLETHORA 3 (AIL6/PLT3) as direct MP-regulated target
genes with a role in flower primordium initiation (228). The authors found that MP acts in
a single input module to directly control the expression of multiple factors that each contribute
to flower formation; this gene regulatory network architecture precludes the identification of
individual factors with roles in flower primordium initiation by genetic means.
LFY was identified as a direct MP target through the discovery of evolutionarily conserved
AuxREs in a key region (16) of the LFY promoter. These AuxREs are bound by MP in vivo (228)
(Figure 6). In mp mutants, inhibition of polar auxin transport and nuclear accumulation of auxininsensitive
Aux/IAA proteins lead to reduced LFY expression, whereas auxin treatment and nuclear
accumulation of MP (even in the presence of a protein synthesis inhibitor) lead to increased LFY
expression (224, 228). Because lfy null mutants do not have defects in flower primordium initiation,
additional direct MP targets may act in parallel with LFY. A candidate approach identified ANT and
AIL6 as being directly MP upregulated at the time of flower primordium initiation (228). Genetic
rescue and genetic enhancer tests demonstrated that LFY, ANT, and AIL6 indeed promote flower
primordium initiation (228). In agreement with these findings, PLT family members were shown
to be important for phyllotaxis (149). Interestingly, both LFY and PLT transcription factors
feedback-regulate auxin accumulation (106, 145, 228). An additional feed-forward loop, in which
ANT/AIL6 directly induce LFY expression, may serve as a signal persistence detector during
flower primordium initiation (227).
lfy ant ail6 triple mutants still initiate rudimental organs but form pin-like inflorescences when
treated with low doses of an auxin transport inhibitor (228), suggesting that additional MP targets
exist that contribute to flower primordium initiation. Recent studies have identified the
genes encoding the polarity regulator and YABBY family transcription factor FIL (56, 174, 183)
and the APETALA 2 (AP2) transcription factor TMO3 [also called CYTOKININ RESPONSE
FACTOR 2 (CRF2)] (155, 177) as direct MP-regulated target genes (Figure 7). Wu et al. (224)
found that MP binds to the promoters of both genes in vivo and that both genes are induced upon
increased nuclear MP accumulation and repressed upon increased nuclear Aux/IAA accumulation.
Several genetic enhancer tests confirmed that FIL plays a role in flower primordium initiation
(224). Whether TMO3/CRF2 also contributes to primordium initiation at the reproductive shoot
apex remains to be determined. The combined findings indicate that during flower primordium
initiation, MP directly activates diverse developmental processes, including floral fate specification
(LFY), cell proliferation and growth (ANT/AIL6), organ polarity (FIL), and cytokinin responses
(ARR7 and -15, AHP6, and TMO3/CRF2) (15, 224, 228, 237). Their activation by MP logically
couples these disparate processes. It is possible that MP induces additional, as yet undiscovered
pathways.
During the last stage of flower primordium differentiation, expression of the pluripotency
gene WUSCHEL (WUS) must be repressed in the center of the flower meristem in order for the
carpel (a part of the female reproductive structure) to form. The floral homeotic gene AGAMOUS
(AG) and Polycomb repression play critical roles in this process (111, 112, 190). Recently, a
genetic enhancer screen linked the repressive ARF ETTIN (ETT)/ARF3 to this pathway. The AG
antagonist AP2, which has nonoverlapping domains of activity with AG in the flower primordium,
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Arabidopsis
Rice
Physcomitrella
Marchantia
Green algae
(e.g., Spirogyra)
ARFAux/IAATIR1/AFB
? ? ?
Figure 7
The evolution of the auxin response pathway, showing the distribution of genes encoding TIR1/AFB,
Aux/IAA, and ARF proteins in published plant genomes for several plant species. These species represent
eudicots (Arabidopsis), monocots (rice), mosses (Physcomitrella), liverworts (Marchantia), and green algae
(Spirogyra, as an example of charophytes). The tree on the left-hand side indicates the divergence order but is
not drawn to scale. Protein abbreviations: ARF, AUXIN RESPONSE FACTOR; Aux/IAA,
AUXIN/INDOLE-3-ACETIC ACID; TIR1/AFB, TRANSPORT INHIBITOR RESISTANT 1/AUXIN
SIGNALING F-BOX.
directly represses ETT. ETT in turn directly represses WUS. The ETT-bound site at the WUS
promoter is close to a region occupied by AG, and ETT binding to WUS (but not to a previously
defined ETT target) was shown to be dependent on AG (110, 112). Hence, AG modulates the
activity of this repressive ARF.
Auxin Response During Gynoecium, Ovule, and Pollen Development
Local auxin maxima and auxin responses play important roles during the formation of the male
and female reproductive structures (reviewed in 83, 103, 163). The female reproductive structure,
the gynoecium, consists of multiple tissue layers with distinct functions, such as the gynophore at
the base, the two valves with the transmitting tract and ovules arising from the valve margins, the
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style, and the stigmatic papillae. Genetic or pharmacological disruption of polar auxin transport
or loss of function of two repressive ARFs (ETT and the closely related ARF4) causes severe
gynoecium patterning defects and female sterility (12, 131, 180, 181). ETT/ARF3 and ARF4
have been proposed to repress expression of the bHLH transcription factors SPATULA and
HECATE (73, 84), although whether this effect is direct is unknown. ETT is required for carpel
valve/ovary formation, and the carpels of the ett arf4 double mutant have polarity defects (131, 180,
181). Likewise, MP plays a role in carpel development, and weak mp mutants form flowers with
carpels that lack carpel margin tissue and ovules (35, 65, 115, 151). These phenotypes are partially
recapitulated by loss of function of the direct MP target LFY (228). A pair of class A ARFs,
ARF6 and ARF8, together regulate both gynoecium and stamen maturation, but their direct
targets remain unknown (128, 223). ARF6 and ARF8 promote expression of HALF FILLED,
a bHLH transcription factor important for transmitting tract development (38). Several of the
transcription factors involved in gynoecium development directly regulate auxin transport and
biosynthesis; these include the bHLH transcription factors SPATULA, INDEHISCENT, and
HECATE as well as a family of B3 transcription factors called NGATHA (69, 126, 179).
MP also plays a role in ovule formation. Defects in ovule formation have been described
for mutants in ANT and double mutants in CUC1 and CUC2, which are known regulators of
organ boundaries (54, 90). A recent study employed conditional cuc1 cuc2 double mutants to
further dissect this pathway and showed that ANT and CUC1/CUC2 act in parallel (65). MP,
ANT, CUC1, and CUC2 were expressed in the placenta before ovules form, and expression of
ANT, CUC1, and CUC2 was reduced in mp mutants; in addition, MP bound to the ANT, CUC1,
and CUC2 promoters (65). These data indicate that MP directs ovule formation by inducing at
least two distinct processes: increased cell proliferation and growth (ANT) in the central region
and establishment of an organ boundary (CUC2) at the periphery of the ovule primordium (65).
Finally, ARF17, a class C repressive ARF, was recently implicated in callose synthesis during pollen
development (229). In arf17 mutants, microspores did not form an exine layer and subsequently
died. The authors traced this defect to reduced callose deposition and reduced expression of
CALLOSE SYNTHASE 5 (CalS5) and provided evidence that ARF17 directly binds to the CalS5
promoter. The arf17 mutant microspore defect is more severe than that in cals5, suggesting that
additional ARF17 targets in this pathway remain unidentified. This evidence suggests that ARF17
plays a role in activating gene expression.
THE EVOLUTION OF THE AUXIN RESPONSE PATHWAY
Auxin is a major regulator of growth and development in land plants, and a key question is when
its response machinery evolved. Phylogenetic analysis of the Aux/IAA and ARF families has shown
that both are represented by multiple members in most plants studied [including, for example,
Arabidopsis (156, 159), poplar (94), and rice (91, 173)], suggesting that these species are capable of
complex auxin responses (Figure 7). The genome sequences of the moss Physcomitrella patens (160)
and the lycophyte Selaginella moellendorffii (5) also encode auxin response systems of considerable
complexity. Functional information on Aux/IAAs and ARFs is lacking for most species. However,
given sequence conservation, auxin responses were likely present in early land plants.
Only recently have studies begun to genetically dissect auxin responses in early-diverging land
plants. Ashton et al. (4) isolated several auxin-resistant P. patens mutants in the 1970s. Following
the sequencing of the P. patens genome (160), Prigge et al. (150) mapped the causal genes to
P. patens auxin response mutants and found that they were caused by mutations in PpAux/IAA
genes. These mutations were analogous to those found in gain-of-function aux/iaa mutants in
Arabidopsis (156) and indeed prevented interaction with the P. patens TIR1/AFB co-orthologs
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(150). Thus, Aux/IAA proteins act in a similar auxin- and TIR1/AFB-dependent manner in a
moss. The P. patens genome encodes multiple TIR1/AFBs, Aux/IAAs, and ARFs (160) and thus
has an elaborate network of auxin response components (Figure 7). However, this species may
have undergone gene losses or duplications and may not be representative of other early-diverging
land plants.
Indeed, recent analysis of the auxin response machinery of the liverwort Marchantia polymorpha
revealed the slimmest system thus far. This species encodes a single TIR1/AFB ortholog, a single
Aux/IAA, and three ARFs (59, 95) (Figure 7). Interestingly, the three ARFs each represent a
different class (A, B, and C), which suggests that the functional diversification of ARFs occurred
early during plant evolution, before the diversification of the Aux/IAA and TIR1/AFB families.
Analyses of M. polymorpha lines with a Cas9-induced mutation in MpARF1 (189), a gain-offunction
mutation in the MpAux/IAA protein (95), or reduced MpAux/IAA expression (artificial
microRNA) (59) all showed a profound involvement of auxin in the growth and development of
this species. Lines with reduced auxin responses showed a variety of defects (60, 95), suggesting
that these responses are essential in multiple aspects of development.
The M. polymorpha auxin response systems appear to operate in much the same way as was
described in Arabidopsis: Aux/IAA and the ARFs can all interact, and ARFs can both homo- and
heterodimerize (95). Whereas an Aux/IAA gain-of-function mutation (95) or ARF knockout (189)
NONTRANSCRIPTIONAL AUXIN RESPONSES
Auxin not only profoundly influences gene transcription, but also affects cell function via nongenomic pathways.
Classical auxin responses include rapid hyperpolarization of the plasma membrane following auxin addition to
protoplasts (10), which is believed to be mediated by activation of the plasma membrane proton pump. Auxininduced
cell growth can also occur very quickly, and this response is intact in TIR1/AFB quadruple receptor mutants
(176). Thus, auxin affects cell growth through a pathway that may not depend on gene regulation. Paciorek et al.
(141) showed that auxin inhibits endocytosis of plasma membrane proteins, again without involving components of
the nuclear response pathway.
For decades, researchers have investigated proteins with auxin-binding properties in plant extracts, with most
of the attention captured by AUXIN-BINDING PROTEIN 1 (ABP1). ABP1 resides primarily in the endoplasmic
reticulum but is also secreted and present in the apoplast (92). Crystal structures confirmed specific auxin binding
but revealed no conformational change upon binding (221). Antibodies raised against ABP1 inhibited auxin-induced
plasma membrane hyperpolarization (186), suggesting that this protein may serve as a receptor for nongenomic
auxin responses. Embryo-lethal phenotypes in T-DNA mutants with an insertion in the ABP1 gene (25) precluded
straightforward genetic analysis of its role, and alternative strategies were used instead. Expression of a fragment of
the anti-ABP1 antibody (104) or an ABP1-antisense RNA fragment (19) induced strong growth defects, suggesting
that ABP1 plays a role in mediating nontranscriptional responses. Likewise, a point mutation in the auxin-binding
pocket derived from targeting induced local lesions in genomes (TILLING) (226) led to defects consistent with
a reduced auxin response. These genetic resources have been used to elucidate a pathway involving ABP1 as an
extracellular receptor, interacting TRANSMEMBRANE KINASE (TMK) receptor kinases, and intracellular ROP
protein activation to alter cytoskeletal properties, cell shape, and endocytosis (26, 165, 225).
Strikingly, though, new knockout alleles generated through genome editing were viable and did not show
obvious phenotypes (66). In addition, Enders et al. (55) showed that the abp1-5 TILLING allele harbors many
other mutations that might condition the auxin response phenotypes in this line. These findings cast doubt on
the importance of ABP1 in mediating important auxin responses. Thus, although it is clear that auxin can trigger
nongenomic responses, the question of whether ABP1 mediates this process is still largely open.
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creates auxin-insensitive plants, ARF overexpression or Aux/IAA downregulation causes auxin
hypersensitivity (59). Finally, MpAux/IAA also appears to act by recruiting the MpTPL protein.
Targeting MpTPL to auxin-responsive genes by fusing it to the DNA-binding domain of MpARF1
induced the same range of phenotypes as the gain-of-function MpAux/IAA mutation (59).
Thus, land plants that diverged as early as M. polymorpha possessed an auxin response system
that displays features of the logic and regulation described in the more complex higher plants.
This finding now allows researchers to address many questions related not only to the wiring and
dynamics of the auxin response system, but also to the evolution of novel specificities, regulation,
and functionality during land plant radiations. Given that M. polymorpha is considered a representative
of the earliest-diverging land plants (18) and encodes three different ARFs, a fundamentally
important question is whether functional auxin responses predated the water-to-land transition.
Largely owing to the small number of available genome sequences for algae, knowledge of algal
auxin function and response is fragmented (44). The availability of additional genome sequences
in the future should help clarify when the auxin response system first evolved.
SUMMARY POINTS
1. A core mechanism for nuclear auxin responses has been identified. This mechanism
involves binding of auxin to both the SCFTIR1/AFB
ubiquitin ligase and its
AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) substrate protein. The subsequent
ubiquitination and degradation of Aux/IAA proteins releases interacting, DNA-binding
AUXIN RESPONSE FACTOR (ARF) transcription factors from inhibition and allows
these to regulate gene transcription.
2. Transcriptional regulation by auxin involves chromatin-level control to sustain the repressed
state and promote the activated state. Repression involves histone deacetylation
upon recruitment of the respective enzyme by the TOPLESS (TPL) corepressor
and the Aux/IAA repressor. Activation requires recruitment of SPLAYED/BRAHMA
(SYD/BRM) chromatin remodelers to the ARF transcription factor.
3. The auxin response machinery has the potential to confer quantitatively different responses
to varying auxin concentrations. All core components are represented by multiple
copies in plant genomes. Interactions among these core factors, each of which has
unique biochemical properties, create a range of possible response outcomes. Whether
cells respond to different auxin concentrations by regulating distinct sets of genes remains
an open question.
4. Despite the brevity of the auxin response pathway, transcriptional, posttranscriptional,
and posttranslational control over core components allows tuning of the pathway by
feedbackregulation,duringdevelopment,orbyotherhormonalorenvironmentalsignals.
5. Specificity in response is critical to the ability to trigger multiple, distinct responses in
different contexts during plant development. Selection of context-specific target genes
is governed by ARF-DNA interactions, which depend on cooperative binding of complex
DNA motifs by ARF dimers. ARF-binding cofactors have been identified that may
mediate specificity in gene regulation. A role for ARFs in controlling access of other
transcription factors to cis-elements in the context of chromatin may also contribute to
specificity.
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6. Several target genes have been isolated that mediate auxin responses in specific developmental
processes. Several of these target genes, often directly regulated by ARF transcription
factors, are expressed only in a specific context, suggesting that auxin indeed
directs local activation of defined subsets of ARF target genes.
7. Analysis of the auxin response system in early-diverging land plants has shown that the
mechanism of signaling has deep roots, going back at least to the liverworts. Simpler auxin
response networks appear to share the same regulatory principles, and the presence of
multiple ARFs in a liverwort suggests that auxin responses may have evolved even earlier.
FUTURE ISSUES
1. The potential for specific auxin responses at different concentrations remains unexplored.
Understanding whether auxin gradients can carry meaningful information across their
length (see sidebar Quantitative Auxin Output: A Plant Morphogen?) will require defining
the sensitivity that cells have to distinguish different auxin concentrations and trigger
distinct sets of genes. Given that cell types differ in their response to auxin, it will be
important to address this question in several well-defined populations of specific cell
types.
2. A structural model exists that explains the binding of ARF transcription factors to a
canonical DNA element in vitro, but in vivo binding preferences have not been systematically
investigated for the ARF family. In vivo binding information will be essential
to determine whether structural properties of ARF proteins themselves are sufficient to
explain binding preferences in vivo. This will also be critical to determine the degree of
specialization of ARFs at the level of target recognition in vivo.
3. Although we now have a better understanding of the activation of gene expression by
auxin sensing, the precise temporal series of events that leads from signal perception to
increased transcription has not yet been elucidated, nor is it known whether all of the
players have been identified. It will be highly informative to dissect this event further and
determine whether it follows the same general logic at different target loci or in different
contexts.
4. A well-grounded model exists to explain auxin-dependent gene activation, but how auxin
represses genes is essentially unknown. Does this process use the same components, or
does it involve other, as yet unknown proteins? Is repression based on inhibitory interactions
between activating and repressing ARFs and/or competition for DNA sites? Are
Aux/IAA proteins involved? A full biochemical and genetic description of gene repression
will shed light on these questions.
5. When did auxin responses evolve, and what were the earliest targets and biological
processes regulated by the hormone? The origin may lie before the evolution of the first
land plants, and exploration of genome information from organisms at the transition
from water to land (e.g., charophytes) will be essential to reconstruct the early evolution
of the pathway.
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6. What does AUXIN-BINDING PROTEIN 1 (ABP1; see sidebar Nontranscriptional
Auxin Responses) do? Is this indeed an extracellular auxin receptor, or do nongenomic
responses require different binding sites? Do nongenomic responses intersect with the
nuclear auxin signaling pathway? And if so, at what station do they do so? Scrutiny of the
role of ABP1 and identification of components mediating fast, nonnuclear auxin effects
will help solve this puzzle.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We apologize to colleagues whose important contributions we could not discuss in this review.
Research on auxin response in D. Weijers’s lab is funded by the Netherlands Organization for
Scientific Research (NWO) (ECHO grant 711.011.002 and VICI grant 865.14.001). Research on
flower development in D. Wagner’s lab is funded by National Science Foundation (NSF) grant
IOS-1257111, and research on chromatin regulation is funded by NSF grant MCB-1243757.
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Ralph J. DeBerardinis
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Roderick L. Beijersbergen, Lodewyk F.A. Wessels,
René Bernards
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Lin He
•	p53: Multiple Facets of a Rubik’s Cube, Yun Zhang,
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•	Deciphering Genetic Intratumor Heterogeneity
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Nicholas McGranahan, Javier Herrero, Charles Swanton
•	Immune-Suppressing Cellular Elements of the Tumor
Microenvironment, Douglas T. Fearon
•	Overcoming On-Target Resistance to Tyrosine Kinase
Inhibitors in Lung Cancer, Ibiayi Dagogo-Jack,
Jeffrey A. Engelman, Alice T. Shaw
•	Apoptosis and Cancer, Anthony Letai
•	Chemical Carcinogenesis Models of Cancer: Back
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•	Extracellular Matrix Remodeling and Stiffening Modulate
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Jennifer L. Leight, Allison P. Drain, Valerie M. Weaver
•	Aneuploidy in Cancer: Seq-ing Answers to Old Questions,
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Kristian Helin, Saverio Minucci
•	Targeted Differentiation Therapy with Mutant IDH Inhibitors:
Early Experiences and Parallels with Other Differentiation
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Annual Review of
Plant Biology
Volume 67, 2016Contents
The Path to Thioredoxin and Redox Regulation in Chloroplasts
Bob B. Buchanan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Learning the Languages of the Chloroplast: Retrograde Signaling
and Beyond
Kai Xun Chan, Su Yin Phua, Peter Crisp, Ryan McQuinn, and Barry J. Pogson p p p p p p p p25
NDH-1 and NDH-2 Plastoquinone Reductases in Oxygenic
Photosynthesis
Gilles Peltier, Eva-Mari Aro, and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p55
Physiological Functions of Cyclic Electron Transport Around
Photosystem I in Sustaining Photosynthesis and Plant Growth
Wataru Yamori and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p81
The Costs of Photorespiration to Food Production Now
and in the Future
Berkley J. Walker, Andy VanLoocke, Carl J. Bernacchi, and Donald R. Ort p p p p p p p p p p p 107
Metabolite Damage and Metabolite Damage Control in Plants
Andrew D. Hanson, Christopher S. Henry, Oliver Fiehn,
and Val´erie de Cr´ecy-Lagard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131
The Regulation of Essential Amino Acid Synthesis and Accumulation
in Plants
Gad Galili, Rachel Amir, and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153
Triacylglycerol Metabolism, Function, and Accumulation in Plant
Vegetative Tissues
Changcheng Xu and John Shanklin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 179
The Plant Polyester Cutin: Biosynthesis, Structure, and Biological
Roles
Eric A. Fich, Nicholas A. Segerson, and Jocelyn K.C. Rose p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207
Biosynthesis of the Plant Cell Wall Matrix Polysaccharide Xyloglucan
Markus Pauly and Kenneth Keegstra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 235
TOR Signaling and Nutrient Sensing
Thomas Dobrenel, Camila Caldana, Johannes Hanson, Christophe Robaglia,
Michel Vincentz, Bruce Veit, and Christian Meyer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
v
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Rapid, Long-Distance Electrical and Calcium Signaling in Plants
Won-Gyu Choi, Richard Hilleary, Sarah J. Swanson, Su-Hwa Kim,
and Simon Gilroy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Endocytosis and Endosomal Trafficking in Plants
Julio Paez Valencia, Kaija Goodman, and Marisa S. Otegui p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 309
Staying Tight: Plasmodesmal Membrane Contact Sites and the
Control of Cell-to-Cell Connectivity in Plants
Jens Tilsner, William Nicolas, Abel Rosado, and Emmanuelle M. Bayer p p p p p p p p p p p p p p p p 337
Pre-Meiotic Anther Development: Cell Fate Specification and
Differentiation
Virginia Walbot and Rachel L. Egger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365
Plant Sex Chromosomes
Deborah Charlesworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 397
Haploidization via Chromosome Elimination: Means and Mechanisms
Takayoshi Ishii, Raheleh Karimi-Ashtiyani, and Andreas Houben p p p p p p p p p p p p p p p p p p p p p p p 421
Mechanisms Used by Plants to Cope with DNA Damage
Zhubing Hu, Toon Cools, and Lieven De Veylder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 439
The Conservation and Function of RNA Secondary Structure in Plants
Lee E. Vandivier, Stephen J. Anderson, Shawn W. Foley, and Brian D. Gregory p p p p p p 463
Toxic Heavy Metal and Metalloid Accumulation in Crop Plants
and Foods
Stephan Clemens and Jian Feng Ma p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
Light-Mediated Hormonal Regulation of Plant Growth
and Development
Mieke de Wit, Vinicius Costa Galv˜ao, and Christian Fankhauser p p p p p p p p p p p p p p p p p p p p p p p 513
Transcriptional Responses to the Auxin Hormone
Dolf Weijers and Doris Wagner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 539
Mapping Transcriptional Networks in Plants: Data-Driven Discovery
of Novel Biological Mechanisms
Allison Gaudinier and Siobhan M. Brady p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 575
The Intracellular Dynamics of Circadian Clocks Reach for the Light
of Ecology and Evolution
Andrew J. Millar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 595
Environmental Control of Root System Biology
Rub´en Rell´an- ´Alvarez, Guillaume Lobet, and Jos´e R. Dinneny p p p p p p p p p p p p p p p p p p p p p p p p p p 619
The Haustorium, a Specialized Invasive Organ in Parasitic Plants
Satoko Yoshida, Songkui Cui, Yasunori Ichihashi, and Ken Shirasu p p p p p p p p p p p p p p p p p p p p p 643
vi Contents
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Antibody Production in Plants and Green Algae
Vidadi Yusibov, Natasha Kushnir, and Stephen J. Streatfield p p p p p p p p p p p p p p p p p p p p p p p p p p p p 669
Perennial Grain and Oilseed Crops
Michael B. Kantar, Catrin E. Tyl, Kevin M. Dorn, Xiaofei Zhang,
Jacob M. Jungers, Joe M. Kaser, Rachel R. Schendel, James O. Eckberg,
Bryan C. Runck, Mirko Bunzel, Nick R. Jordan, Robert M. Stupar,
M. David Marks, James A. Anderson, Gregg A. Johnson, Craig C. Sheaffer,
Tonya C. Schoenfuss, Baraem Ismail, George E. Heimpel,
and Donald L. Wyse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 703
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant
Contents vii
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