© 2015 Authors; published by Portland Press Limited Essays Biochem. (2015) 58,165-181: doi: 10.1042/BSE0580165
Comparison of plant hormone signalling systems
Antoine Larrieu and Teva Vernoux1
Laboratoire de Reproduction et Développement des Plantes, CNRS, INRA, ENS Lyon, UCBL, Universitě de Lyon, 69364 Lyon, France
Abstract
Plant growth and development are controlled by nine structurally distinct small molecules termed phytohormones. Over the last 20 years, the molecular basis of their signal transduction, from receptors to transcription factors, has been dissected using mainly Arabidopsis thaliana and rice as model systems. Phytohormones can be broadly classified into two distinct groups on the basis of whether the subcellular localization of their receptors is in the cytoplasm or nucleus, and hence soluble, or membrane-bound, and hence insoluble. Soluble receptors, which control the responses to auxin, jasmonates, gibberellins, strigo-lactones and salicylic acid, signal either directly or indirectly via the destruction of regulatory proteins. Responses to abscisic acid are primarily mediated by soluble receptors that indirectly regulate the phosphorylation of targeted proteins. Insoluble receptors, which control the responses to cytokinins, brassinosteroids and ethylene, transduce their signal through protein phosphorylation. This chapter provides a comparison of the different components of these signalling systems, and discusses the similarities and differences between them.
Key words:
abscisic acid, auxin, brassinosteroids, cytokinins, ethylene, F-Box E3 ubiquitin ligases, gibberellins, jasmonic acid, phosphorylation cascade, phytohormones, salicylic acid, SCF complex, serine/threonine kinases, strigolactones, ubiquitination.
1To whom correspondence should be addressed (email teva.vernoux@ens-lyon.fr).
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Introduction
Plant growth and development depend on numerous signals that include endogenous molecules, such as peptides and metabolites, as well as external cues. These signals converge on small signalling molecules called phytohormones, affecting their metabolism, transport and/or signalling, and rendering them central regulators of the plant life cycle. To date, nine families of phytohormones for which receptors are known (Table 1) have been identified in plants.
Hormone signalling involves two distinct steps. First, the signal is recognized in the target cells or tissues by a receptor. Secondly, upon binding of the ligand to the receptor, a signal is transmitted downstream through a variable number of intermediates towards the final targets (hereafter termed effectors). Importantly, hormone signals not only switch genes on and off but also control other non-transcriptional responses, such as ion pump activity and clathrin-mediated endocytotic recycling. However, in this chapter we shall focus exclusively on transcriptional responses, as these have been extensively described in the literature.
Phytohormone receptors are either localized at the plasma membrane (PM), endoplasmic reticulum (ER) or endosomes, and are therefore insoluble, or are localized in the cytoplasm and/or the nucleus, and are therefore soluble (Table 1). This has important implications for the way that phytohormones transduce their signal towards the effectors. Membrane-bound receptors of cytokinins (CKs) and brassinosteroids (BRs) act as multimers that trigger a phosphorylation cascade through histidine kinases (in the case of CKs) or serine/threonine kinases (in the case of BRs). Cytosolic and/or nuclear receptors of auxin, jasmonates (JAs), gib-berellins (GAs) and strigolactones (SLs) mediate their responses through the controlled degradation of proteins by the proteasome, either through a 'molecular glue' mechanism (in the case of auxin and JAs) or via an adaptor protein (in the case of GAs and SLs). Salicylic acid (SA) signalling remains elusive, although its soluble receptors have been recently identified. Abscisic acid (ABA) signalling is controlled by a family of nucleo cytoplasmic receptors that trigger the phosphorylation of a family of transcription factors (TFs), which is a unique situation among soluble receptors in plants. Finally, ethylene signalling offers a mixture of phosphotransfers, controlled by its insoluble ER-localized receptors, and protein degradation, controlled by several E3 ubiquitin ligases. In the following account we highlight the similarities and differences between the various hormonal signalling pathways.
Auxin, jasmonates (JAs), gibberellins (GAs), strigolactones (SLs), abscisic acid (ABA) and salicylic acid (SA): signalling through cytoplasmic and/or nuclear receptors
Soluble receptors of auxin and JAs are localized in the nucleus, whereas GA and SL receptors are also found in the cytoplasm. Their signalling pathways rely on protein polyubiquitination by the SCF (Skp-Cullin-F-box) complex (see below for details) and their subsequent degradation by the proteasome. ABA signal transduction starts in the cytoplasm or the nucleus, and leads to the phosphorylation of a family of TFs. SA signalling remains elusive despite the recent identification of its receptors, which are found in both the cytoplasm and the nucleus.
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Table 1. The receptors, intermediates, effectors, signalling systems and molecular structures of the nine phytohormones
The table summarizes all of the different components and the different systems used by phytohormones to transduce their canonical transcriptional responses. Note that there are other receptors described, notably for auxin and ABA signalling. All of the gene names indicated are from Arabidopsis except for SL signalling, for which they come from rice. The effector of SL signalling BES1 was identified in Arabidopsis.
Component Auxin
Jasmonates Gibberellins   Strigolactones Salicylic Abscisic (JAs) (GAs) (SLs) acid(SA)    acid (ABA)
Ethylene (ET)    Cytokinins Brassino-
(CKs) steroids (BRs)
Receptor(s)     TIR1, COI1 AFB1-AFB5
Receptor        F-Box F-Box gene family
Subcellular      Nucleus Nucleus localization of the receptor(s)
Other known    ABP1 (PM, receptors ER) (subcellular localization)
Intermediates   Aux/IAAst JAZsf
GID1a-GID1c     DWARF 14
Hormone- a/ß-Hydrolase sensitive lipase
Nucleus and Nucleus cytoplasm
SLY, SNE, DELLA
DWARF 53
NPR1, PYR1-PYR14 ETR1 and
NPR3 and ETR2, EIN4,
NPR4* ERS1 and ERS2
BTB/POZ START domain- Histidine kinase
domain containing protein
Nucleus and   Nucleus and cytoplasm cytoplasm
GTGs (PM), CHLH
(chloroplast)
NPR1 PP2Cs, SnRK2.2,
SnRK2.3 and SnRK2.6
ER
CTR1, EIN2
AHK2-AHK4
Histidine kinase
Plasma membrane and ER
BRI1, BRL1 and BRL3
Serine/threonine protein kinases
Plasma
membrane and early endosomes
AHP1-AHP6
BAK1t, BKI1, BSK1/CDG1, BSU1, BIN2
(Continued)
Table 1. The receptors, intermediates, effectors, signalling systems and molecular structures of the nine phytohormones (Continued)
Component	Auxin	Jasmonates	Gibberellins	Strigolactones	Salicylic	Abscisic acid	Ethylene (ET)	Cytokinins	Brassino-
		(JAs)	(GAs)	(SLs)	acid (SA)	(ABA)		(CKs)	steroids (BRs)
Effectors	ARFs	bHLH* TFs (MYC2, ...)	PIFs,...	BES1	bZIP* TFs (TGA, ...)	bZIP* TFs(ABI3, ABI5, ...)	EIN3, EILs	A and B type ARRs	BZR1, BES1
Signalling	SCF-	SCF-	SCF-mediated	SCF-mediated	Protein	Phosphorylation	Phosphorylation	Phosphoryl-	Phosphoryl-
system	mediated	mediated	protein	protein	degradation		and SCF-	ation	ation
	protein	protein	degradation	degradation	(?)		mediated protein		
	degradation	degradation					degradation		
Molecular					Q		H2C=CH,		
structure	H							CH.HM^Nr JL J H	
Name	lndole-3-acetic acid	(+)-7-/so-jasmonoyl-L-isoleucine	Gibberellic acid A3	(+)-Strigol	Salicylic acid	Abscisic acid	Ethylene	Zeatin	Brassinolide
*The role of the SA receptors NPR1, NPR3 and NPR4 appears to be important, but their function as primary SA receptors needs to be confirmed, intermediates in signalling pathways that directly interact with the phytohormone, and hence that are considered to be co-receptors. *Not all proteins belonging to these gene families, but only a specific subfamily, participate in hormonal responses.
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Functions of the SCF complex
The SCF complex controls the polyubiquitination of proteins in eukaryotes. Ubiquitin is a polypeptide (consisting of 76 amino acids) that is added to proteins post-translationally and functions as a marker for protein degradation, translocation and many other processes. Ubiquitination is a process whereby three classes of enzymes act in concert to add one or several ubiquitin moieties to a target protein. It involves an El ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin-ligase enzyme that recruits the substrate for ubiquitination. F-Box proteins are a class of E3 ubiquitin ligases that work in a complex termed the SCF complex. This complex is composed of four core subunits, namely SKP1, CULLIN1, a variable F-Box E3 ubiquitin ligase and RBX1 (Figure 1). The complex forms a horseshoe-shaped structure that brings a substrate specifically recruited by the F-Box subunit into the vicinity of an E2 ubiquitin-conjugating enzyme, recruited by the RBX1 subunit. The substrate is then rapidly polyubiquitinated and degraded by the proteasome. The Arabidopsis genome encodes more than 700 F-Box proteins, which can theoretically bind a similar number of distinct protein families. It plays pivotal roles during multiple cellular processes, but notably in hormonal signalling, including auxin, JA, GA, SL and ethylene signalling.
Auxin and Jasmonate (JA) signalling
Auxin and JAs transduce their signal in a remarkably similar way. The bioactive molecules are indole-3-acetic acid (IAA) for auxin, and JA conjugated to the amino acid isoleucine (+)-7-/so-jasmonoyl-l-isoleucine or JA-Ile for JA (Table 1). These small compounds act as a 'molecular glue' between pairs of proteins that function as co-receptors, which are on one side of the F-Box E3 ubiquitin ligases, and on the other side are specific proteins that act as transcriptional repressors [1,2] (Figure 1). In Arabidopsis there are six nuclear-localized F-box co-receptors for auxin in the core pathway (see Table 1 and the Discussion for alternative receptors), termed TRANSPORT-INHIBITOR RESPONSE 1 (TIR1) and AUXIN SIGNALLING F-BOX PROTEIN 1 to 5 (AFB1-AFB5), whereas there is only one receptor for jasmonates, CORONATINE-INSENSITIVE 1 (COI1). Auxin repressors are encoded by a protein family called the Aux/IAAs, whereas JA repressors are members of the JASMONATE ZIM DOMAIN (JAZ) gene family [3,4]. Aux/IAAs and JAZs interact with their co-receptors through a specific domain, and this interaction is greatly stabilized in the presence of auxin and JA-Ile respectively. The term 'molecular glue' has been coined to describe the function of these hormones in mediating protein-protein interactions, and the term 'co-receptor' is used to describe the co-operative binding of the hormone in the presence of both the F-Box E3 ligase and the transcriptional repressor, as has been clearly demonstrated for auxin [5].
Aux/IAA proteins repress TFs that belong to the auxin-response factors (ARFs) family, whereas JAZ proteins repress multiple TFs that notably belong to the basic helix-loop-helix (bHLH) family (such as MYC2), but also include other TFs [6,7]. ARF proteins fall into two categories, namely those that activate and those that repress transcription [8]. In the absence of the hormones, Aux/IAA and JAZ repressors are stabilized and repress transcription through the recruitment of a transcriptional repressor called TOPLESS (TPL) [9]. Although Aux/IAAs and some JAZ proteins can recruit TPL directly through an EAR motif, several JAZ proteins require another protein termed NOVEL INTERACTOR OF JAZ (NINJA) [10]. In the presence of the
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Figure 1. The SCF complex and signal transductions of auxin, JA, GA and SL
(A) Schematic representation of the SCF complex. (B) Signal transduction of auxin and JA starts in the nucleus. In the absence of these hormones, transcriptional responses are repressed through the recruitment of the TOPLESS complex in the promoter of regulated genes. In the presence of the hormones, repressors of auxin and JA signalling are degraded by the SCF complex (only the F-Box protein is shown), and transcriptional responses are triggered (either activation or repression of regulated genes). The intermediates and effectors of auxin signalling are shown in white, and those of JA signalling are shown in yellow. (C) Signal transduction of GA and SL starts in the nucleus and/or cytoplasm. In the absence of these hormones, transcriptional responses are repressed directly by DELLA proteins (GA) or through the recruitment of the TOPLESS complex (SL) in the promoter of regulated genes. In the presence of the hormones, repressors of GA and SL signalling are degraded by the SCF complex (only the F-Box protein is shown due to space limitations), and transcriptional responses are triggered (either activation or repression of regulated genes). The intermediates and effectors of GA signalling are shown in white, and those of SL are shown in yellow. All of the gene names indicated are from Arabidopsis, except for SL signalling, for which they come from rice. The effector of SL signalling, BES1, was identified in Arabidopsis.
hormones, Aux/IAAs and JAZ proteins are rapidly polyubiquitinated by the SCFTIR1/AFB1~AFB5 and SCFcon complexes respectively, and subsequently degraded by the 26S proteasome [11] (Figure 1).
Gibberellin (GA) and Strigolactone (SL) signalling
Whereas auxin and JA receptors are F-Box proteins themselves, the receptors and the F-Box proteins in GA and SL signalling are uncoupled (Figure 1). Their signalling pathways involve additional protein-protein interactions, notably through adaptor proteins, that lead to the rapid
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degradation of targeted proteins via the SCF complex. Most of the original research on GA and SL signalling has been undertaken on rice, and therefore the gene names cited in this section are from rice. However, the names of homologous genes in Arabidopsis are also provided for consistency.
The rice GA receptor, GA-INSENSITIVE DWARF 1 (GID1), belongs to the family of hormone-sensitive lipases (HSLs), whereas the only SL receptor identified so far, namely DWARF14 (D14), belongs to the family of 0c/(3 hydrolases [12,13]. In Arabidopsis there are three homologous GID1 genes (GIDla, GIDlb and GIDlc) and at least one SL-receptor-homologous gene (also termed DWARF 14).
Structural studies of the binding of GA to GID1 have shown that GA triggers a conformational change which allows the direct interaction of GID1 with a DELLA protein termed SLENDER RICE 1 (SLR1), a repressor of GA signalling [14]. The GID1-GA-SLR1 complex can interact with the F-Box protein GID2, which triggers the polyubiquitination of SLR1 by the SCFGID2 complex and its degradation by the proteasome (Figure 1). In Arabidopsis there are five homologous genes for SLR, termed REPRESSOR OF gal-3 (RGA), GA-INSENSITIVE (GAI) and RGA-LIKE 1-3 (RGL1-RGL3), and two homologous genes for GID2 termed SLEEPY (SLY) and SNEEZY (SNE). Following the degradation of DELLA proteins, several effectors of GA signalling, such as the phytochrome-interacting factors (PIFs), can bind DNA and modulate the expression of GA target genes [15,16].
There are still large gaps in our understanding of SL signalling. However, in the same manner as GA, SL binding to its receptor D14 triggers a conformational change that allows the SL-D14 complex to interact with the F-Box E3 ligase DWARF3 (D3) and with a repressor of SL signalling that resides in the nucleus, DWARF53 (D53) [13]. This interaction leads to the polyubiquitination of D53 by the SCFD3 complex and its degradation by the proteasome, which in turn leads to the induction of SL-responsive genes (Figure 1) [17]. In Arabidopsis there eight homologous genes for SLR, termed SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE 2-8 (SMXL2-SMXL8), and there is one homologous gene for D3 termed MORE AXILLARY BRANCHES (MAX2). Effectors of SL signalling have so far remained elusive. However, BR-INSENSITIVE 1 EMS-SUPPRESSOR 1 (BES1), which also regulates BR responses, appears to be targeted for degradation by an SCFD3 complex [18].
Abscisic acid (ABA) signalling
ABA perception and signalling are complex, and combine not one but several families of receptors, some of which are membrane bound, whereas others are present in the cytosol or nucleus. Here we shall only focus on the core pathway (for alternative receptors, see Table 1), which is mediated by a family of soluble receptors known as PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR)/PYR-LIKE (PYL) proteins, which will be referred to as PYRs hereafter (Figure 2). In the absence of ABA, a clade of protein phosphatases 2C (PP2Cs) maintain SNF1-related kinases (SnRKs) 2.2, 2.3 and 2.6 in a dephosphorylated and hence inactive state [19]. Structural studies have shown that ABA binding to PYRs is achieved through a 'gate-latch-lock' mechanism, and is increased in the presence of PP2Cs [20,21]. However, this mechanism is different from those described for auxin and JA signalling, as there is no direct contact of PP2Cs with the hormone, and hence these are not ABA co-receptors. In the presence of ABA, PYR proteins interact with and inactivate PP2C2, which releases SnRKs to phosphorylate basic leucine zipper (bZIP) TFs, ion pumps and other effectors of ABA signalling [22] (Figure 2).
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Phosphorylation on a serine or threonine or tyrosine residue Dephosphorylation on a serine or threonine or tyrosine residue
Change in gene expression
Figure 2. Signal transduction of ABA
ABA signalling starts in the cytoplasm or the nucleus. In the absence of this hormone, the level of phosphorylated effectors of ABA signalling (belonging to the bZIP family of TFs) is low, thus hindering hormonal responses. In the presence of the hormone, the receptors sequester PP2Cs phosphatase, which allows several members of a family of serine/threonine kinases, the SnRKs (SnRK2.2, SnRK2.3 and SnRK2.6) to phosphorylate effectors of ABA signalling. This in turn triggers transcriptional responses (either activation or repression of regulated genes). All of the gene names indicated are from Arabidopsis.
Salicylic acid (SA) signalling
Important advances have recently been made in our understanding of SA signalling. However, its signal transduction remains poorly understood. In two studies, three related proteins, NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1 1, 3 and 4 (NPR1, NPR3 and NPR4), were shown to be SA receptors in Arabidopsis [23,24]. However, it is unclear whether all three proteins actually bind SA. In the first study [23], it was shown that NPR3 and NPR4 control the levels of NPR1 in the nucleus through the ubiquitin-proteas-ome system in an SA-dependent manner. In the second study [24], NPR1 was shown to bind SA (NPR3 and NPR4 were not tested). Some results suggest that the binding of SA to NPR1 triggers a conformational change that allows NPR1 to bind DNA and regulate gene expression.
There are still many unanswered questions about how SA controls its responses, and further research is needed to elucidate whether or not the two models coexist. Notably, NPR1-mediated induction of SA responses is delayed after SA treatments, and therefore NPR1-, NPR3- and NPR4-mediated signalling appear to act as secondary responses. For this reason, no representation of its signalling system is shown, as at the time of writing no clear picture
Key:
Phosphate on a serine or threonine o: tyrosine residue
ABA
Repression
Transcription factor binding site
r
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has emerged from the literature. However, SA signalling logic is possibly unique among phyto-hormone signalling systems.
Cytokinins (CKs), brassinosteroids (BRs) and ethylene: signalling through transmembrane receptors
CK and BR receptors belong to two different classes of receptor kinases. Histidine kinases control CK signalling, whereas serine/threonine kinases control BR signalling. Both phyto-hormones trigger a reversible cascade of phosphorylation from a membrane, either the ER or the PM respectively. The cascade ends up in the nucleus where effectors of each pathway regulate the expression of target genes. Ethylene has a complex multi-layered signalling pathway, which relies on protein phosphorylation and also on the controlled degradation of specific proteins, which, as for auxin, JAs, GAs and SLs, is controlled by several F-Box proteins.
Cytokinin (CK) signalling
In Arabidopsis, CKs are perceived by a family of two-component histidine kinase receptors [ARABIDOPSIS HISTIDINE KINASES (AHKs) 2, 3 and 4] that are primarily localized at the ER but also found at the PM [25-27] (Figure 3). Upon binding of a bioactive CK, AHKs autophosphorylate the conserved histidine and aspartate residues in their intracellular domain, and transfer it to a conserved histidine residue on ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE (AHP) proteins [28]. Another AHK, CYTOKININ-INDEPENDENT 1 (CKI1), also acts as an upstream regulator of the pathway and of AHP phosphorylation, but most probably in a CK-independent manner [29]. AHP1-AHP5 have the conserved histidine residue and act as positive regulators of CK signalling, whereas AHP6 lacks it, and acts as a negative regulator of CK signalling [30]. AHPs shuttle between the cytoplasm and the nucleus upon phosphorylation. In the nucleus they phosphorylate B-type ARABIDOPSIS RESPONSE REGULATORS (ARRs). Once phosphorylated, B-type ARRs control the expression of cytokinin-responsive genes that include the A-type ARRs, which are also phosphorylated by the AHPs and provide negative feedback on CK signalling [31] (Figure 3).
Brassinosteroid (BR) signalling
BR signal transduction involves a complex phosphorylation/dephosphorylation cascade (Figure 4). At first, BRs are perceived by a family of three leucine-rich repeat (LRR) receptorlike kinases that are localized at the cell surface. BRASSINOSTEROID-INSENSITIVE 1 (BRI1), the main BR receptor in Arabidopsis, and its homologous genes BRI1-LIKE1 and BRI1-LIKE 3 (BRL1 and BRL3), can bind brassinolide (BL), the most bioactive BR, together with one of their co-receptors, BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), BAK1-LIKE 1 (BKK1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (SERK1) [32-34]. As was seen in the example of auxin and JA, brassinolide acts as a 'molecular glue' that brings the receptor and co-receptor together [32]. Binding of BL stimulates the phosphorylation and membrane detachment of BRI1 KINASE INHIBITORl (BKI1), which activates the
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AHK2-4
CKI1
ER Membrane, Plasma membrane
Key:
Translocation
"1
Phosphorylation on a histidina or aspartate residue
Phosphate on a hislidirift or aspartate residue
Repression Transcription factor binding site
ck
Change in gene expression
Figure 3. Signal transduction of CK
CK signalling starts from the plasma membrane or ER membrane. In the presence of the hormone, a phosphorylation cascade is activated by the receptors. CKI1 is able to promote CK responses, but it appears to be unable to bind bioactive CK. In the presence of CK, AHPs are phosphorylated, and this allows them to shuttle from the cytoplasm to the nucleus where they phosphorylate effectors of CK signalling, namely the B-type ARRs, which results in the triggering of transcriptional responses (either activation or repression of regulated genes). Among the genes so regulated, A-type ARRs, which can also be phosphorylated by the AHPs, provide direct feedback on CK responses. All of the gene names indicated are from Arabidopsis.
frans-phosphorylation of the receptor and co-receptor [35]. This allows BRI1 to interact with and phosphorylate two families of membrane-anchored kinases related to BR-SIGNALLING KINASE 1 (BSK1) and CONSTITUTIVE DIFFERENTIAL GROWTH1 (CDGls). Phosphorylated BSKs and CDGs phosphorylate a family of cytoplasmic phosphatases related to BRI1-SUPPRESSOR 1 (BSU1) [36]. Phosphorylated BSU1 inactivates BRASSINOSTEROID-INSENSITIVE2 (BIN2), a soluble serine/threonine kinase, by dephosphorylating a conserved tyrosine residue. The level of phosphorylated BIN2 controls the activity of BR signalling effectors, the TFs BRASSINAZOLE-RESISTANT 1 and 2 (BZR1 and BZR2) [37]. Phosphorylated BIN2 phosphorylates the BZRs, which inactivates them. Conversely, inactivation of BIN2 by BSU1 triggers the dephosphorylation of BZRs by a clade of protein phosphatases 2A (PP2As). Non-phosphorylated BZRs interact with other TFs to regulate the expression of BR target genes [37] (Figure 4).
Ethylene (ET) signalling
There are five ethylene receptors encoded in the Arabidopsis genome, namely ETHYLENE-RESPONSE 1 and 2 (ETR1 and ETR2), ETHYLENE-RESPONSE SENSOR 1 and 2 (ERS1 and
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Plasma membrane Early endosomes
—I BRI1 BRL1.3
BAK1	BSKs
BKK1	CDGs
SERK1	
BRI1 bsks BRL1,3   ■     BKK1 CDGs
i/SERK1 «
Key:
Phosphate on a serine or threonine or tyrosine residue Repression
Transcription factor binding site
BRs
Phosphorylation On a serine or threonine or tyrosine residue
□©phosphorylation on a serine or threonine or tyrosine residue
Change in gene expression
Figure 4. Signal transduction of BR
BR signalling starts from the plasma membrane or the membrane of early endosomes. In the absence of the hormone, the receptor BRI1 is kept inactive by BKI1. In the presence of the hormone, a phosphorylation cascade is activated by the receptors. The cascade leads to the inactivation of BIN2, which increases the proportion of unphosphorylated BES1 and BZR1 (collectively referred to as BZRs). Unphosphorylated BZRs trigger transcriptional responses (either activation or repression of regulated genes). All of the gene names indicated are from Arabidopsis.
ESR2) and ETHYLENE-INSENSITIVE 4 (EIN4), which are all localized in the ER membrane [38] (Figure 5). Like CK receptors, they share a strong homology with bacterial two-component histidine kinases. However, it is unclear whether the kinase activity is essential for their function. The receptors interact with CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), a serine/threonine kinase that in the absence of ethylene phosphorylates the C-terminal end of EIN2, which is also
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ETR1 -2
ERS1-2 -ethylene EIN4
ETR1-2 ERS 1-2 EIN4
■+ ethylene
^-^^/ Translocation
Phosphate on a %    serine or threonine or tyrosine residue
^_    Transcription factor binding site
• Ubiquitin
Phosphorylation on a serine or threonine or tyrosine residue
Ethylene
Change in gene expression
Figure 5. Signal transduction of ethylene
Ethylene signalling starts from the ER membrane. In the absence of the hormone, the receptors are constitutively active, leading to the phosphorylation of EIN2, which is the central regulator of ethylene signalling. Phosphorylated EIN2 is rapidly degraded, thus hindering ethylene responses. In the presence of ethylene, EIN2 is not phosphorylated, and this leads to the proteolytic cleavage of its C-terminus, which is translocated to the nucleus where it stabilizes effectors of ethylene signalling that belong to the EIN3/EILs family of TFs. This in turn triggers transcriptional responses (either activation or repression of regulated genes). All of the gene names indicated are from Arabidopsis.
located on the ER membrane [39]. In response to an ethylene signal, CTR1 is inactivated, which reduces EIN2 phosphorylation. This triggers the cleavage of EIN2, and its C-terminus is translocated to the nucleus, where it interacts with and participates in the stabilization of key effectors of ethylene signalling, namely EIN3 and EIN3-LIKE 1 to 3 (EIL1-3) [39-41].
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There are two additional levels of ethylene signalling (Figure 5). First, EIN2 protein stability is regulated by two F-Box proteins, namely EIN2-TARGETING PROTEIN 1 and 2 (ETP1 and ETP2), which trigger EIN2 degradation in the absence of ethylene [42]. Secondly, EIN3 and EIL1 protein levels are also regulated by two F-Box proteins, EIN3-BINDING F-BOX 1 and 2 (EBF1 and EBF2), which also trigger EIN3 and EIL1 degradation in the absence of ethylene [43,44]. The complexity underlying these interactions is still largely a mystery. However, they probably allow the integration of multiple signals to fine-tune ethylene responses.
Discussion
As we have seen, hormones in plants use three main strategies to transduce their signals to their effectors, namely the control of protein degradation, protein phosphorylation, or a combination of both. The end result is usually reprogramming of the repertoire of genes expressed by the target cell. A key aspect of phytohormones is that both individually and in various combinations they control very diverse developmental processes.
This is primarily achieved by the redundancy at each step of their signalling pathways. For instance, CK signalling includes four receptors (the AHKs and CKI1), six intermediates (the AHPs) and 21 effectors (the ten A-type and 11 B-type ARRs). As different homologues have different biochemical properties and expression profiles, the available combinations of receptors and intermediates, of intermediates and effectors, and of effectors between them allow for a vast range of possible responses. In addition, hormonal cross-talk allows these responses to be fine-tuned. For example, the signalling pathway of both GA and BR converges on the PIF TFs, which are master regulators in the control of cell elongation and growth [45].
In this chapter we have focused on signalling systems that lead to changes in gene expression. However, there are numerous examples of hormonal signals triggering non-transcriptional responses. For instance, auxin can bind a PM- and ER-localized protein, termed AUXIN-BINDING PROTEIN 1 (ABP1). ABP1 has historically been regarded as the regulator of non-transcriptional auxin responses, and notably involved in clathrin-medi-ated endocytosis. However, the link between ABP1 and downstream responses has remained elusive despite its crucial function, as demonstrated by the embryonic lethality of abpl loss-of-function mutants. A recent study may have identified receptor-like kinase transmembrane kinases, TRANSMEMBRANE KINASE 1-4 (TMK1-TMK4), as the missing links signalling downstream of ABP1 [46]. Future research will determine the impact of ABP1 on auxin responses, and notably on their interaction with the TIR1/AFB1-AFB5 pathway [47], although the importance of this alternative auxin receptor has recently been challenged [48].
Overall, it can be noted that signalling between protein degradation and protein phosphorylation will have a direct impact on the dynamics of the response. Indeed, protein phosphorylations are reversible, thus allowing their signalling pathways to be swiftly turned on and off. In contrast, protein degradation is permanent and requires de novo translation to shut down a signalling pathway which, in terms of time and energy cost, is significant compared with protein phosphorylation.
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Conclusions
An understanding of phytohormone signalling is essential for advances in both fundamental and applied research. Novel elite crop varieties can be selected based on particular traits related to hormone signalling. This is exemplified by the Green Revolution, which took place between the 1970s and the 1990s, and saw the emergence of novel crop cultivars that had much improved yields, which were later shown to be primarily linked to altered GA signalling.
The recent identification of the receptors for SLs and SA, which are involved in the regulation of shoot branching and the response to pathogens respectively, is a very exciting development [23,24,49]. In view of the much reduced increments in wheat production resulting from plateauing yields, these discoveries offer promising routes for exploration by plant breeders.
Finally, the deciphering of hormone signalling allows the design of specific tools for studying signal transduction, such as the novel hormone biosensors reported in the literature [50]. These will enable a much better understanding of the fundamental processes that govern hormone signalling and plant development.
Summary
• There are nine small signalling molecules that have been described in plants and for which receptors are known. These are termed phytohormones.
• Phytohormone receptors that are soluble, and hence localized in the cytoplasm and/or the nucleus, include auxin, jasmonates, gibberellins, strigolac-tones, salicylic acid and abscisic acid. Those that are insoluble, and hence membrane-bound, include brassinosteroids, cytokinins and ethylene.
• Their signalling pathways can be classified as protein-phosphorylation-dependent or protein-degradation-dependent, or a combination of both.
• Protein degradation through the SCF complex plays a key role in regulating the transcriptional responses to auxin, jasmonates, gibberellins, strigolac-tones and ethylene.
• Abscisic acid promotes the phosphorylation of bZIP TFs, which allows their binding to DNA and the regulation of target genes.
• Salicylic acid signalling remains poorly understood. However, it is expected that important discoveries will be made in the near future, as its receptors have recently been identified.
• Histidine kinase receptors control the responses to cytokinins and ethylene. The kinase activity of the receptor is required for cytokinin signalling, but it is unclear whether this is also the case for ethylene signalling.
• A phosphorylation cascade controls plant responses to brassinosteroids and cytokinins.
• Ethylene is the only phytohormone that negatively regulates its receptor, which is otherwise constitutively activated in the absence of the signal.
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