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ScienceDirect Strigolactone signalling: standing on the shoulders of DWARFs Tom Bennett and Ottoline Leyser Strigolactones are an ancient and major class of endogenous plant growth regulators. Although only recently identified, rapid progress has been made in understanding strigolactone biology, including the identification of a signalling pathway involving DWARF14 a/b-fold proteins, the SCFMAX2 ubiquitin ligase and SMAX1-LIKE (SMXL) family of chaperonin-like proteins. Several rapid effects of strigolactone signalling have also been identified, including endocytosis of the PINFORMED1 (PIN1) auxin efflux carrier and transcript accumulation of the BRANCHED1 (BRC1) transcription factor. Here we assess our current knowledge of strigolactone signalling, and discuss how increased understanding of the cell biology of the system can help to resolve some of the current uncertainties in the field. Addresses Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge CB2 1LR, UK Corresponding author: Leyser, Ottoline ([email protected])
Current Opinion in Plant Biology 2014, 22:7–13 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.08.001 1369-5266/Published by Elsevier Ltd.
Introduction Despite their relatively recent arrival on the plant signalling scene, strigolactones (SLs) are an ancient class of hormones that regulate many aspects of plant development. As intrinsic growth regulators, SLs have been implicated in the control of shoot branching [1], stature [1], root growth [2] and root hair elongation [3], in particular with respect to responses to environmental stimuli such as phosphate availability [4,5]. Furthermore, SLs exuded from roots are key exogenous signals in establishing arbuscular mycorrhizal symbioses [6], and also promote the germination of several parasitic plant species [reviewed in 7]. Here, we review some of the remarkable progress that has recently been made in understanding SL biology. We first examine our current understanding of the mechanism and targets of SL signalling. We then discuss these data with regard to a cell www.sciencedirect.com
biological framework, and assess how this corresponds to the apparent targets and functions of SLs.
The canonical strigolactone signalling pathway Recent progress in understanding SL biology has led to the emergence of what could be defined as the canonical signalling pathway for SL (Figure 1a). There is now a substantial body of data regarding the function of the DWARF14 (D14) class of a/b-fold proteins as probable SL receptors from rice [8,9,10], Arabidopsis [11,12,13] and petunia [14] (Table 1). Although there is still considerable uncertainty about exactly how D14 proteins operate [reviewed in 15,16], they can bind to the SL analogue GR24 and d14 mutants in several species are insensitive to SLs [10,11,12,14], strongly supporting the hypothesis that D14 is a receptor for SLs. Moreover, in the tissues where it has been examined, there is very high phenotypic overlap between d14 mutants and SL synthesis mutants [11], suggesting that D14 is the receptor for the majority of developmental SL responses. D14 appears to interact with the second component of the pathway, the MAX2 class of F-box protein [14,17,18] (Table 1), although the precise details of this interaction are somewhat hazy, particularly with respect to its SL dependence [reviewed in 15]. Like so many other hormone signalling components in plants, MAX2 proteins act as part of an SCF-type ubiquitin ligase that is assumed to regulate ubiquitination and degradation of target proteins [19,20]. Excitingly, recent work in rice has revealed what appear to be clear targets for SL-induced MAX2-D14-dependent protein degradation, namely members of the SMXL protein family [17,18,21] (Table 1). In rice, deletion of a specific 5 amino acid motif within the SMXL protein, DWARF53 (D53), results in a dominant SL-resistant increased branching phenotype, while knock-downs of D53 are able effectively to suppress the phenotype of dwarf3 (d3; a mutant in the rice orthologue of MAX2 (Table 1)), and d14 mutants [17,18]. D53 appears to interact physically with both D14 and D3. The D53 protein is rapidly degraded in the presence of GR24, and this is blocked in the dominant D53 mutant [17,18]. These data, and particularly the precise phenotypic match and phenotype suppression caused by the d53 mutants, suggest that D53 class proteins are the primary target of SL signalling, at least for shoot branching, and hence that D53 acts as a constitutive negative regulator of SL responses. Further work is needed to confirm exactly Current Opinion in Plant Biology 2014, 22:7–13
8 Cell biology
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the SMXL family [21; reviewed in 23,24]. Therefore in their essential details, these signalling pathways seem identical (Figure 2). Although other targets for MAX2 have been proposed (see below), the congruence of these two signalling pathways suggests that SMXL family members are the main, canonical targets of MAX2-dependent hormonal signalling.
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(a) A hypothetical model of strigolactone signalling. Binding of SL (yellow) to D14 a/b-fold proteins causes a conformational change (dotted yellow line) that permits tight interaction of D14 (blue) and D53 chaperonin-like proteins (green) independently of MAX2 class F-box proteins (orange) [17,18]. Although D14 can bind to MAX2 independently of D53 and SL [17,18], it seems that only the D14/D53 complex forms a substrate for ubiquitination by SCFMAX2 (purple). Both D14 and D53 are probably ubiquitinated (red) and degraded as a result of the interaction with SCFMAX2 [13,17,18]. (b) Open issues in the cell biology of strigolactone signalling. D14 (blue) and MAX2 (orange) are both present in the cytoplasm and nucleus, while D53 (green) seems to be primarily nuclear; the active site(s) of SL signalling is unclear. D53 might inhibit the activity of TPR-family co-repressor proteins (pink) in the nucleus, and thus indirectly modulate transcription [17,18]; however, evidence for major transcriptional changes upon SL addition is lacking. Alternatively or additionally, D53 might inhibit the endocytosis of PIN1 protein (yellow) from the plasma membrane, possibly by modulating activity of a cytoplasmic CTLH complex (grey), though this is at odds with the predominately nuclear localization of D53. Non-cell autonomous effects of SL signalling might be mediated through cell-to-cell movement of D14 [13] or by downstream modulation of auxin (IAA) transport between cells [34,36].
which SL-dependent processes are regulated by D53 (and the D53 class proteins SMXL6, SMXL7 and SMXL8 in Arabidopsis), but given the phenotypic match, it is likely that the majority of SL signalling acts through degradation of D53 proteins. The canonical nature of this signalling pathway is supported by the closely related, and indeed overlapping signalling pathway for smoke-derived ‘karrikin’ signals, which probably mimic an as-yet-unknown endogenous signal [22]. Karrikin signalling in Arabidopsis acts through KAI2 — a close relative of D14 — and MAX2. It is negatively regulated by SMAX1, the prototypical member of Current Opinion in Plant Biology 2014, 22:7–13
Targets of strigolactone signalling Although a core mechanism of SL signalling now seems clear based on genetic data, there is still significant controversy over the targets, both proximal and ultimate, of SL signalling. Biochemical approaches have suggested two other classes of proteins as direct targets of MAX2, namely the brassinosteroid activated transcription factor BES1 (and its relatives) [25] and the DELLA-class transcription factors which are the primary target of gibberellin signalling [26]. However, the genetic data to support these interactions are either absent in the case of DELLAs [27] or limited and based on highly pleiotropic mutants in the case of BES1 [25]. Furthermore, since there is limited phenotypic overlap between SL and brassinosteroid or gibberellin mutants in general, it seems unlikely that SL signalling affects these other processes as its principal target. Although MAX2-dependent degradation of these proteins may allow crosstalk between these different hormonal pathways, which is of course likely to be of considerable importance, based on currently available evidence it seems unlikely that they are central components of SL signalling. As regards more distal targets of SL signalling, there has been a pervasive assumption in the field that the ultimate effects of SL signalling are mediated through changes in transcription. However, it is important to note that there is relatively little effect of SL addition on the transcriptome. The abundance of very few transcripts is robustly altered in a fast and direct manner after SL treatment [28; reviewed in 24]. This also argues against BES1 and DELLAs as major targets of SL signalling, since significant transcriptional changes would be expected in response to degradation of these well-characterized transcription factors, unless they are only degraded in small subset of the total BES1/DELLA expressing cells. By some distance the best candidate for transcriptional regulation by SL is BRC1 (encoding a TCP transcription factor), which in pea is upregulated in a fast and translation-independent manner after SL treatment [29,30]. Furthermore, Arabidopsis brc1 mutants have increased and SL-resistant branching [30,31]. However, FINECULM1 (FC1), a BRC1 homologue from rice that regulates shoot branching, is not SL responsive. Furthermore, double mutant analysis shows that the effects of BRC1 and SL signalling on branching are at least partially independent in rice [32] and Arabidopsis [13], and are qualitatively different in pea [30]. Changes in BRC1 www.sciencedirect.com
Strigolactone signalling Bennett and Leyser 9
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reviewed in 15]. SL has also been implicated in the regulation of PIN2 polarity and actin behaviour in the Arabidopsis root, although here the effect appears to be non-cell autonomous [36]. Remodelling of auxin transport processes, as a general mechanism, certainly has the potential to explain the global effects of SL signalling. However, beyond the specific examples cited here, there is currently little evidence that changes in PIN endocytosis underlie the broader effects of SL signalling. Indeed, there is no a priori reason for all SL effects to be mediated through a unified downstream mechanism. The current uncertainty surrounding downstream effects in SL signalling demonstrates that although we now have a genetically well-defined core SL signalling pathway, a priority is to develop a deeper understanding of the cell biology of this system. In the next sections, we will examine the current gaps in our knowledge, and how these may be addressed.
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Possible scheme for evolution of SL and karrikin signalling pathways. In charophyte algae, endogenous karrikin-like molecules (‘KL’, brown), and possibly SLs, signal via KAI2 proteins (light blue) and unknown effectors (red). In mosses (and other basal land plants), KAI2 signalling has become associated with SCFMAX2 (orange/purple) and leads to the degradation of SMXL proteins (light green). SLs mostly signal via the ancient MAX2-independent mechanism, but still could still plausibly interact with the newly evolved KAI2-MAX2-SMXL signalling system as well. KL might signal via both mechanisms. In angiosperms, subfunctionalization or neo-functionalization of KAI2-like and SMXL proteins, followed by co-evolution, leads to establishment of SCFMAX2dependent SL signalling via D14–D53 (dark blue/dark green). The specificity of KAI2-SMAX1 signalling may also have increased such that endogenous SLs cannot signal via KAI2 [44,45], and karrikins (KAR) and KL cannot signal via D14. It is likely that KAI2 cannot interact with D53 (and likewise that D14 cannot interact with SMAX1). However, the MAX2-binding domain of the KAI2/SMAX1 and D14/D53 pairs is not changed, such that no sub-functionalization of MAX2 occurs. MAX2independent effects of SL in Arabidopsis [2] also suggest the persistence of the ancient ‘non-canonical’ SL signalling pathway.
expression are thus only a partial explanation for the effect of SL on branching. Moreover, since BRC1 is not broadly expressed throughout the plant [31], changes in BRC1 expression seem unlikely to explain more than a small subset of the global responses to SL. In summary, while it certainly cannot be ruled out, there is little evidence that the major effects of SL signalling are mediated primarily through changes in transcription.
Protein partners for D53 The rice D53 protein contains three possible EAR motifs, which are also found in proteins that interact with TPR transcriptional co-repressors, and it has thus been suggested that D53 regulates transcription of SL response genes in a TPR-dependent manner (Figure 1b) [17,18]. Although D53 appears to bind weakly to some of the TPR proteins [17], there is currently no evidence that its EAR motifs are actually relevant for SL signalling. Moreover only one of these domains is found in SMXL6/SMXL7/ SMXL8 [18], while SMAX1 class proteins contain a divergent type of EAR motif (Table 2). An important priority for understanding SL signalling is therefore to establish the importance of the EAR motifs in D53 class proteins. Even if they are functional, EAR motifs are not exclusively associated with TPR-mediated transcriptional repression, but rather mediate interactions with proteins containing corresponding CTLH domains [37]. In Arabidopsis, several CTLH-domain containing proteins are found in a large cytoplasmic complex that regulates cytoskeletal organization [38], and in yeast CTLH-domain proteins are involved in the targeted endocytosis of fructose-1,6-bisphosphatase [39] (Figure 1b). The presence of an EAR motif in D53 proteins thus in no way predicates that they must function in transcription, and it is plausible that they function in regulating endocytosis. Either way, an important priority for understanding downstream events in SL signalling will be to identify protein partners for D53 via biochemical and genetic approaches.
Sub-cellular localization of SL signalling Removal of the auxin efflux carrier PIN1 from the plasma membrane has been suggested as a fast and direct outcome of SL signalling in Arabidopsis [33,34] (nonmutually exclusive with transcriptional responses), and has been proposed to regulate bud outgrowth via an auxin transport canalization-dependent mechanism [34,35; www.sciencedirect.com
The different distal targets proposed for SL signalling tend to imply distinct sub-cellular localizations for the SL signalling complex (Figure 1b), and thus understanding where and how SL signalling occurs within the cell may help to distinguish between these possibilities, although the current data are somewhat unhelpful in this regard. Current Opinion in Plant Biology 2014, 22:7–13
10 Cell biology
Table 1 Nomenclature and relationship of proteins described in this study. Several classes of proteins participate in SL and karrikin signalling, listed at the left; each class is named after its founding member. Members of these classes have widely varying names between species, summarized in the body of the table. When discussing SL signalling in general terms, we have used the name of the class to represent all members. When we refer to a particular species, we have used the name of the protein from that species. Protein type a/b-fold F-box
Class D14 KAI2 MAX2
Arabidopsis (At) D14 KAI2 MAX2
Rice (Os) D14 D14L D3
Pea (Ps) Petunia (Ph) DAD2 RMS4
MAX2a MAX2b
HSP101/chaperonin-like D53 SMXL6 SMXL7 MXL8 D53 SMAX1 SMAX1 [Os08g15230] TCP transcription factor TB1 BRC1 BRC2 FC1 BRC1
D14 proteins appear to be present in both the cytoplasm and nucleus [13,18], as do MAX2 proteins [18,20], while published data from protoplasts and root cells suggest a primarily nuclear localization for D53 proteins [17,18]. If SL signalling does occur in the nucleus, then it is somewhat remarkable that none of these proteins contain strong nuclear localization signals, and the relevance of cytoplasmic D14 and MAX2 is unclear. Manipulating the sub-cellular localization of SL signalling components by targeting them to specific cellular compartments could therefore be highly informative in establishing exactly where in the cell SL signalling is required for different processes. A complementary approach would be to use fluorescently labelled versions of these proteins, to analyse where in the cell they actually interact with
Function SL receptor Karrikin receptor Signalling intermediate Target of SL signalling Target of Karrikin signalling? Putative downstream target of SL signalling
each other. Bimolecular fluorescence complementation suggests that D53 and D14 from rice interact in the nucleus, but this has only been demonstrated in a heterologous tobacco system [18]. It will also be important to test the localization of each protein in genetic backgrounds mutant for the others, to assess how these proteins affect each other’s sub-cellular localization.
Cell-autonomy of SL signalling Another aspect worth examining is the expression pattern and cell autonomy of SL signalling. The published expression patterns of SL signalling components do not always match well with SL-triggered responses. For example, while MAX2 is expressed in vascular-associated cells in most organs in Arabidopsis, it does not appear to
Table 2
Rice Rice Arabidopsis Arabidopsis Arabidopsis Poplar Poplar Tomato Tomato Arabidopsis Arabidopsis Poplar Poplar Tomato Rice Physcomitrella Physcomitrella
Name/ accession number D53 D53-like SMXL6 SMXL7 SMXL8 Potri.001G252500 Potri.008G069100 Solyc09g055230 Solyc06g051460 SMAX1 SMXL2 Potri.018G097300 Potri.006G175200 Solyc07g006540 Os08g15230 Pp1s248_7V6 Pp1s30_156V6
1st position
2nd position
3rd position
LVLNLQ LVLNLQ FQGMVA YLETPK ~~~~~~ VPRLKQ CILSKQ PAQSLE SSIFLS LRQPLQ ~~~~~~ PRQPFQ PHQPFQ LRQPLQ PRPPVE ~~~~~~ ~~~~~~
LDLNLQ LDLNLQ VDFKSQ VDFGAE VDLGAA LDLGSH ADLSAQ VDLGSS VDLSLQ LGS~~~ LGS~~~ LGS~~~ LGS~~~ LGS~~~ FGGDSR LGFADG FGDLGY
FDLNLP FDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP FDLNQA FDLNEA FDLNEA FDLNEA FDLNEA LDLNLA LDLNLA LDLNLS
SMAX1 clade
Species
D53 clade
EAR (ethylene responsive element binding factor-associated amphiphilic repression) motifs and SMXL proteins. Three potential LxLxLtype EAR motifs have been identified in D53 from rice [17,18], but two of these are not conserved in D53 homologues from other species, suggesting they are either spurious or of limited relevance to the general function of proteins in the D53 sub-clade. The third EAR motif is well conserved across this sub-clade of the SMXL family, suggesting it is of functional relevance. EAR motifs are found in SMAX1 from Arabidopsis and other eudicot members of the SMAX1 clade, but they are of the divergent FDLN-type. The significance of this is not clear, and the rice SMAX1 homologue has an LxLxL-type motif in the same position. EAR motifs are also found in third position in SMXL homologues from the basal land plant Physcomitrella patens; these proteins do not fall into either the D53 or SMAX1 sub-clade.
Notes: Red = Leucine residues that might form part of an EAR motif, Blue = Asparagine or Aspartate residues that might form part of an EAR motif.
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Strigolactone signalling Bennett and Leyser 11
be well expressed in the root epidermis or root hair cells [20,40], both of which are sensitive to GR24 (and therefore potentially SL) [3,36]. In the case of root hair elongation, MAX2 expression in the endodermis is sufficient to control developmental effects in the epidermis, suggesting noncell autonomous outputs for SL signalling [36,41], but how this operates, and whether it is sufficient for all putative epidermal effects of SL is unclear. D14 is expressed in a broadly similar pattern to MAX2 in Arabidopsis, but distinct differences between D14 promoter and protein fusions suggest that D14 might have a degree of mobility (though it cannot act graft-transmissably) [13]. However, the consequences of this movement and whether it relates to non-cell autonomous signalling is also unclear. No detailed expression pattern for SMXL6/SMXL7/SMXL8 has been reported yet, although D53 in rice appears to be expressed in an analogous pattern to D14 and MAX2 [18]. Ultimately, more sensitive tools are required to assess exactly where SL signalling components are expressed and where they function, and whether very low expression levels are sufficient for meaningful activity. It is also likely that some of the putative non-cell autonomous effects of SLs could result from changes in auxin distribution downstream of SL signalling [36,41], and care should therefore be exercised when interpreting apparent SL responses in non-vascular-associated tissues.
Evolution and non-canonical strigolactone signalling One of the most fascinating aspects of SL signalling is its evolution, since the canonical SL and karrikin signalling pathways clearly share an evolutionary origin. The currently available evidence points to the karrikin pathway being the more ancient of the two, since the members of the D14/KAI2 clade in basal land plants and algae are unambiguously similar to KAI2 [11,42]. Members of the D14 and D53 sub-clades have so far only been detected in vascular plants [11,18], suggesting that the canonical SL signalling pathway is a relatively recent innovation, probably arising by partial duplication of the karrikin pathway, followed by sub-functionalization or neo-functionalization (and presumably co-evolution) of the new D14 and D53 pair (Figure 2). However, SLs are present in, and GR24 can regulate development in algae and basal land plants [42,43], clearly implying the existence of more ancient, non-canonical SL signalling mechanisms. One very plausible possibility is that SL signals through KAI2-SMAX1 in early land plants, due to lower specificity in the binding pocket of KAI2 (Figure 2). In Arabidopsis, endogenously occurring SLs do not seem to be able to signal via KAI2 [44,45], but it is possible that after the duplication event that created the pair, both KAI2 and D14 have evolved greater binding specificity than is present in the KAI2-like protein from which they originated (Figure 2). However, it appears that MAX2 has www.sciencedirect.com
evolved specifically in land plants [46], and so this hypothesis is insufficient to explain SL signalling in algae, which must be MAX2-independent. Furthermore, there also appear to be MAX2-independent effects of GR24 in Arabidopsis [2], and SL signalling in moss also may not act through MAX2 [47]. An intriguing hypothesis is therefore that there is a more ancient SL signalling mechanism, possibly involving a KAI2 and/or D14 family member, which has been maintained throughout land plant evolution even though largely superseded by the emergence of canonical D14-MAX2-D53-dependent signalling (Figure 2).
Conclusions The 6 year period since the identification of SLs as endogenous growth regulators has seen an explosion in interest in their biology. The establishment of a canonical signalling pathway in that short time is a remarkable achievement, and seems a good point to take stock of what we now know. Despite — or perhaps because of — the rapid progress, there remain many gaps, contradictions or uncertainties in our understanding of SL biology. What is now required is a degree of consolidation and careful analysis integrating genetic, cell and molecular biological approaches with biochemistry and structural biology to establish more firmly the fundamentals of SL signalling.
Acknowledgements Our research is funded by the Gatsby Foundation (GAT3272C) and the European Research Council (No. 294514 – EnCoDe).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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22. Flematti GR, Waters MT, Scaffidi A, Merritt DJ, Ghisalberti EL, Dixon KW, Smith SM: Karrikin and cyanohydrin smoke signals provide clues to new endogenous plant signaling compounds. Mol Plant 2013, 6:29-37.
10. Kagiyama M, Hirano Y, Mori T, Kim SY, Kyozuka J, Seto Y, Yamaguchi S, Hakoshima T: Structures of D14 and D14L in the strigolactone and karrikin signaling pathways. Genes Cells 2013, 18:147-160. One of several recent papers describing the crystal structure of D14/KAI2 a/b hydrolases, and providing insights into the likely receptor function of these proteins. 11. Waters MT, Nelson DC, Scaffidi A, Flematti GR, Sun YK, Dixon KW, Smith SM: Specialisation within the DWARF14 protein family confers distinct responses to karrikins and strigolactones in Arabidopsis. Development 2012, 139:1285-1295. This report elegantly demonstrates that two members of the same a/b hydrolase family mediate responses to strigolactones and karrikins respectively, further establishing the idea that these signalling pathways share an evolutionary origin. 12. Zhao LH, Zhou XE, Wu ZS, Yi W, Xu Y, Li S, Xu TH, Liu Y, Chen RZ, Kovach A et al.: Crystal structures of two phytohormone signal-transducing a/b hydrolases: karrikin-signaling KAI2 and strigolactone-signaling DWARF14. Cell Res 2013, 23:436-439. One of several recent papers describing the crystal structure of D14/KAI2 a/b hydrolases, providing insights into the likely receptor function of these proteins. 13. Chevalier F, Nieminen K, Sa´nchez-Ferrero JC, Rodrı´guez ML, Chagoyen M, Hardtke CS, Cubas P: Strigolactone promotes degradation of DWARF14, an a/b hydrolase essential for strigolactone signaling in Arabidopsis. Plant Cell 2014, 26:1134-1150. An interesting data set providing several important insights into SL signalling, such as the degradation of D14 protein and it is broad cellular localization. 14. Hamiaux C, Drummond RS, Janssen BJ, Ledger SE, Cooney JM, Newcomb RD, Snowden KC: DAD2 is an a/b hydrolase likely to be involved in the perception of the plant branching hormone, strigolactone. Curr Biol 2012, 22:2032-2036. Describes the cloning of a D14 ortholog from Petunia, DAD2, underlining the conserved nature of the SL signalling pathway. The paper also provides insights into the structure of DAD2 and demonstrates the likely hydrolysis of SL during the course of signalling. 15. Waldie T, McCulloch H, Leyser O: Strigolactones and the control of plant development: lessons from shoot branching. Plant J 2014. Feb 25. 16. Seto Y, Yamaguchi S: Strigolactone biosynthesis and perception. Curr Opin Plant Biol 2014, 21C:1-6. 17. Jiang L, Liu X, Xiong G, Liu H, Chen F, Wang L, Meng X, Liu G, Yu H, Yuan Y et al.: DWARF 53 acts as a repressor of strigolactone signalling in rice. Nature 2013, 504:401-405. One of a pair of recent reports showing that D53 is a major target of MAX2-dependent SL signalling in rice, required for increased tillering in the absence of SL or SL signalling components. 18. Zhou F, Lin Q, Zhu L, Ren Y, Zhou K, Shabek N, Wu F, Mao H, Dong W, Gan L et al.: D14-SCF(D3)-dependent degradation of D53 regulates strigolactone signalling. Nature 2013, 504:406-410. One of a pair of recent reports showing that D53 is a major target of MAX2-dependent SL signalling in rice, required for increased tillering in the absence of SL or SL signalling components. Current Opinion in Plant Biology 2014, 22:7–13
23. Waters MT, Scaffidi A, Sun YK, Flematti GR, Smith SM: The karrikin response system of Arabidopsis. Plant J 2014. Jan 16. 24. Smith SM, Li J: Signalling and responses to strigolactones and karrikins. Curr Opin Plant Biol 2014, 21C:23-29. 25. Wang Y, Sun S, Zhu W, Jia K, Yang H, Wang X: Strigolactone/ MAX2-induced degradation of brassinosteroid transcriptional effector BES1 regulates shoot branching. Dev Cell 2013, 27:681-688. 26. Nakamura H, Xue YL, Miyakawa T, Hou F, Qin HM, Fukui K, Shi X, Ito E, Ito S, Park SH et al.: Molecular mechanism of strigolactone perception by DWARF14. Nat Commun 2013, 4:2613. 27. de Saint Germain A, Ligerot Y, Dun EA, Pillot JP, Ross JJ, Beveridge CA, Rameau C: Strigolactones stimulate internode elongation independently of gibberellins. Plant Physiol 2013, 163:1012-1025. 28. Mashiguchi K, Sasaki E, Shimada Y, Nagae M, Ueno K, Nakano T, Yoneyama K, Suzuki Y, Asami T: Feedback-regulation of strigolactone biosynthetic genes and strigolactoneregulated genes in Arabidopsis. Biosci Biotechnol Biochem 2009, 73:2460-2465. 29. Dun EA, de Saint Germain A, Rameau C, Beveridge CA: Antagonistic action of strigolactone and cytokinin in bud outgrowth control. Plant Physiol 2012, 158:487-498. 30. Braun N, de Saint Germain A, Pillot JP, Boutet-Mercey S, Dalmais M, Antoniadi I, Li X, Maia-Grondard A, Le Signor C, Bouteiller N et al.: The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching. Plant Physiol 2012, 158:225-238. 31. Aguilar-Martı´nez JA, Poza-Carrio´n C, Cubas P: Arabidopsis BRANCHED1 acts as an integrator of branching signals within axillary buds. Plant Cell 2007, 19:458-472. 32. Minakuchi K, Kameoka H, Yasuno N, Umehara M, Luo L, Kobayashi K, Hanada A, Ueno K, Asami T, Yamaguchi S, Kyozuka J: FINE CULM1 (FC1) works downstream of strigolactones to inhibit the outgrowth of axillary buds in rice. Plant Cell Physiol 2010, 51:1127-1135. 33. Crawford S, Shinohara N, Sieberer T, Williamson L, George G, Hepworth J, Mu¨ller D, Domagalska MA, Leyser O: Strigolactones enhance competition between shoot branches by dampening auxin transport. Development 2010, 137:2905-2913. 34. Shinohara N, Taylor C, Leyser O: Strigolactone can promote or inhibit shoot branching by triggering rapid depletion of the auxin efflux protein PIN1 from the plasma membrane. PLoS Biol 2013, 11:e1001474. This report follows up previous studies [30,32], and demonstrates that SL rapidly mediates endocytosis of PIN1 protein, independently of new translation. 35. Prusinkiewicz P, Crawford S, Smith RS, Ljung K, Bennett T, Ongaro V, Leyser O: Control of bud activation by an auxin transport switch. Proc Natl Acad Sci U S A 2009, 106:17431-17436. 36. Pandya-Kumar N, Shema R, Kumar M, Mayzlish-Gati E, Levy D, Zemach H, Belausov E, Wininger S, Abu-Abied M, Kapulnik Y, Koltai H: Strigolactone analog GR24 triggers changes in PIN2 polarity, vesicle trafficking and actin filament architecture. New Phytol 2014. Feb 25. www.sciencedirect.com
Strigolactone signalling Bennett and Leyser 13
This report shows that SL treatment affects PIN2 expression and localization in root epidermal cells, and suggests that SL may also affect actin remodelling. 37. Szemenyei H, Hannon M, Long JA: TOPLESS mediates auxindependent transcriptional repression during Arabidopsis embryogenesis. Science 2008, 319:1384-1386. 38. Kobayashi N, Yang J, Ueda A, Suzuki T, Tomaru K, Takeno M, Okuda K, Ishigatsubo Y: RanBPM, Muskelin, p48EMLP, p44CTLH, and the armadillo-repeat proteins ARMC8alpha and ARMC8beta are components of the CTLH complex. Gene 2007, 396:236-247. 39. Giardina BJ, Dunton D, Chiang HL: Vid28 protein is required for the association of vacuole import and degradation (Vid) vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction. J Biol Chem 2013, 288:11636-11648. 40. Brady SM, Orlando DA, Lee JY, Wang JY, Koch J, Dinneny JR, Mace D, Ohler U, Benfey PN: A high-resolution root spatiotemporal map reveals dominant expression patterns. Science 2007, 318:801-806. 41. Koren D, Resnick N, Mayzlish Gati E, Belausov E, Weininger S, Kapulnik Y, Koltai H: Strigolactone signaling in the endodermis is sufficient to restore root responses and involves SHORT HYPOCOTYL 2 (SHY2) activity. New Phytol 2013, 198:866-874. 42. Delaux PM, Xie X, Timme RE, Puech-Pages V, Dunand C, Lecompte E, Delwiche CF, Yoneyama K, Be´card G, Se´jalonDelmas N: Origin of strigolactones in the green lineage. New Phytol 2012, 195:857-871. A timely investigation into the distribution of SLs and SL-related genes across the plant kingdom, showing that SLs appear to be very ancient
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signals. This study also provides a clear framework for understanding the apparently recent evolution of canonical SL signalling. 43. Proust H, Hoffmann B, Xie X, Yoneyama K, Schaefer DG, Yoneyama K, Nogue´ F, Rameau C: Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens. Development 2011, 138:1531-1539. 44. Scaffidi A, Waters MT, Ghisalberti EL, Dixon KW, Flematti GR, Smith SM: Carlactone-independent seedling morphogenesis in Arabidopsis. Plant J 2013, 76:1-9. A thorough study demonstrating that although the SL analogue GR24 can activate KAI2-dependent signalling, endogenous SLs derived from carlactone can only signal through D14. 45. Scaffidi A, Waters M, Sun YK, Skelton BW, Dixon KW, Ghisalberti EL, Flematti G, Smith S: Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis. Plant Physiol 2014 May 7. A follow up to [41], examining how natural and non-naturally occurring stereoisomers of SLs act in plants. D14 displays stereoselectivity towards naturally occurring SLs, while non-naturally occurring enantiomers signal mainly through KAI2, thus showing that stereochemistry is a crucial aspect of SL signalling specificity. 46. Challis RJ, Hepworth J, Mouchel C, Waites R, Leyser O: A role for more axillary growth1 (MAX1) in evolutionary diversity in strigolactone signalling upstream of MAX2. Plant Physiol 2013, 161:1885-1902. 47. de Saint Germain A, Bonhomme S, Boyer FD, Rameau C: Novel insights into strigolactone distribution and signalling. Curr Opin Plant Biol 2013, 16:583-589.
Current Opinion in Plant Biology 2014, 22:7–13
ScienceDirect Strigolactone signalling: standing on the shoulders of DWARFs Tom Bennett and Ottoline Leyser Strigolactones are an ancient and major class of endogenous plant growth regulators. Although only recently identified, rapid progress has been made in understanding strigolactone biology, including the identification of a signalling pathway involving DWARF14 a/b-fold proteins, the SCFMAX2 ubiquitin ligase and SMAX1-LIKE (SMXL) family of chaperonin-like proteins. Several rapid effects of strigolactone signalling have also been identified, including endocytosis of the PINFORMED1 (PIN1) auxin efflux carrier and transcript accumulation of the BRANCHED1 (BRC1) transcription factor. Here we assess our current knowledge of strigolactone signalling, and discuss how increased understanding of the cell biology of the system can help to resolve some of the current uncertainties in the field. Addresses Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge CB2 1LR, UK Corresponding author: Leyser, Ottoline ([email protected])
Current Opinion in Plant Biology 2014, 22:7–13 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.08.001 1369-5266/Published by Elsevier Ltd.
Introduction Despite their relatively recent arrival on the plant signalling scene, strigolactones (SLs) are an ancient class of hormones that regulate many aspects of plant development. As intrinsic growth regulators, SLs have been implicated in the control of shoot branching [1], stature [1], root growth [2] and root hair elongation [3], in particular with respect to responses to environmental stimuli such as phosphate availability [4,5]. Furthermore, SLs exuded from roots are key exogenous signals in establishing arbuscular mycorrhizal symbioses [6], and also promote the germination of several parasitic plant species [reviewed in 7]. Here, we review some of the remarkable progress that has recently been made in understanding SL biology. We first examine our current understanding of the mechanism and targets of SL signalling. We then discuss these data with regard to a cell www.sciencedirect.com
biological framework, and assess how this corresponds to the apparent targets and functions of SLs.
The canonical strigolactone signalling pathway Recent progress in understanding SL biology has led to the emergence of what could be defined as the canonical signalling pathway for SL (Figure 1a). There is now a substantial body of data regarding the function of the DWARF14 (D14) class of a/b-fold proteins as probable SL receptors from rice [8,9,10], Arabidopsis [11,12,13] and petunia [14] (Table 1). Although there is still considerable uncertainty about exactly how D14 proteins operate [reviewed in 15,16], they can bind to the SL analogue GR24 and d14 mutants in several species are insensitive to SLs [10,11,12,14], strongly supporting the hypothesis that D14 is a receptor for SLs. Moreover, in the tissues where it has been examined, there is very high phenotypic overlap between d14 mutants and SL synthesis mutants [11], suggesting that D14 is the receptor for the majority of developmental SL responses. D14 appears to interact with the second component of the pathway, the MAX2 class of F-box protein [14,17,18] (Table 1), although the precise details of this interaction are somewhat hazy, particularly with respect to its SL dependence [reviewed in 15]. Like so many other hormone signalling components in plants, MAX2 proteins act as part of an SCF-type ubiquitin ligase that is assumed to regulate ubiquitination and degradation of target proteins [19,20]. Excitingly, recent work in rice has revealed what appear to be clear targets for SL-induced MAX2-D14-dependent protein degradation, namely members of the SMXL protein family [17,18,21] (Table 1). In rice, deletion of a specific 5 amino acid motif within the SMXL protein, DWARF53 (D53), results in a dominant SL-resistant increased branching phenotype, while knock-downs of D53 are able effectively to suppress the phenotype of dwarf3 (d3; a mutant in the rice orthologue of MAX2 (Table 1)), and d14 mutants [17,18]. D53 appears to interact physically with both D14 and D3. The D53 protein is rapidly degraded in the presence of GR24, and this is blocked in the dominant D53 mutant [17,18]. These data, and particularly the precise phenotypic match and phenotype suppression caused by the d53 mutants, suggest that D53 class proteins are the primary target of SL signalling, at least for shoot branching, and hence that D53 acts as a constitutive negative regulator of SL responses. Further work is needed to confirm exactly Current Opinion in Plant Biology 2014, 22:7–13
8 Cell biology
Figure 1
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the SMXL family [21; reviewed in 23,24]. Therefore in their essential details, these signalling pathways seem identical (Figure 2). Although other targets for MAX2 have been proposed (see below), the congruence of these two signalling pathways suggests that SMXL family members are the main, canonical targets of MAX2-dependent hormonal signalling.
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(a) A hypothetical model of strigolactone signalling. Binding of SL (yellow) to D14 a/b-fold proteins causes a conformational change (dotted yellow line) that permits tight interaction of D14 (blue) and D53 chaperonin-like proteins (green) independently of MAX2 class F-box proteins (orange) [17,18]. Although D14 can bind to MAX2 independently of D53 and SL [17,18], it seems that only the D14/D53 complex forms a substrate for ubiquitination by SCFMAX2 (purple). Both D14 and D53 are probably ubiquitinated (red) and degraded as a result of the interaction with SCFMAX2 [13,17,18]. (b) Open issues in the cell biology of strigolactone signalling. D14 (blue) and MAX2 (orange) are both present in the cytoplasm and nucleus, while D53 (green) seems to be primarily nuclear; the active site(s) of SL signalling is unclear. D53 might inhibit the activity of TPR-family co-repressor proteins (pink) in the nucleus, and thus indirectly modulate transcription [17,18]; however, evidence for major transcriptional changes upon SL addition is lacking. Alternatively or additionally, D53 might inhibit the endocytosis of PIN1 protein (yellow) from the plasma membrane, possibly by modulating activity of a cytoplasmic CTLH complex (grey), though this is at odds with the predominately nuclear localization of D53. Non-cell autonomous effects of SL signalling might be mediated through cell-to-cell movement of D14 [13] or by downstream modulation of auxin (IAA) transport between cells [34,36].
which SL-dependent processes are regulated by D53 (and the D53 class proteins SMXL6, SMXL7 and SMXL8 in Arabidopsis), but given the phenotypic match, it is likely that the majority of SL signalling acts through degradation of D53 proteins. The canonical nature of this signalling pathway is supported by the closely related, and indeed overlapping signalling pathway for smoke-derived ‘karrikin’ signals, which probably mimic an as-yet-unknown endogenous signal [22]. Karrikin signalling in Arabidopsis acts through KAI2 — a close relative of D14 — and MAX2. It is negatively regulated by SMAX1, the prototypical member of Current Opinion in Plant Biology 2014, 22:7–13
Targets of strigolactone signalling Although a core mechanism of SL signalling now seems clear based on genetic data, there is still significant controversy over the targets, both proximal and ultimate, of SL signalling. Biochemical approaches have suggested two other classes of proteins as direct targets of MAX2, namely the brassinosteroid activated transcription factor BES1 (and its relatives) [25] and the DELLA-class transcription factors which are the primary target of gibberellin signalling [26]. However, the genetic data to support these interactions are either absent in the case of DELLAs [27] or limited and based on highly pleiotropic mutants in the case of BES1 [25]. Furthermore, since there is limited phenotypic overlap between SL and brassinosteroid or gibberellin mutants in general, it seems unlikely that SL signalling affects these other processes as its principal target. Although MAX2-dependent degradation of these proteins may allow crosstalk between these different hormonal pathways, which is of course likely to be of considerable importance, based on currently available evidence it seems unlikely that they are central components of SL signalling. As regards more distal targets of SL signalling, there has been a pervasive assumption in the field that the ultimate effects of SL signalling are mediated through changes in transcription. However, it is important to note that there is relatively little effect of SL addition on the transcriptome. The abundance of very few transcripts is robustly altered in a fast and direct manner after SL treatment [28; reviewed in 24]. This also argues against BES1 and DELLAs as major targets of SL signalling, since significant transcriptional changes would be expected in response to degradation of these well-characterized transcription factors, unless they are only degraded in small subset of the total BES1/DELLA expressing cells. By some distance the best candidate for transcriptional regulation by SL is BRC1 (encoding a TCP transcription factor), which in pea is upregulated in a fast and translation-independent manner after SL treatment [29,30]. Furthermore, Arabidopsis brc1 mutants have increased and SL-resistant branching [30,31]. However, FINECULM1 (FC1), a BRC1 homologue from rice that regulates shoot branching, is not SL responsive. Furthermore, double mutant analysis shows that the effects of BRC1 and SL signalling on branching are at least partially independent in rice [32] and Arabidopsis [13], and are qualitatively different in pea [30]. Changes in BRC1 www.sciencedirect.com
Strigolactone signalling Bennett and Leyser 9
Figure 2
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reviewed in 15]. SL has also been implicated in the regulation of PIN2 polarity and actin behaviour in the Arabidopsis root, although here the effect appears to be non-cell autonomous [36]. Remodelling of auxin transport processes, as a general mechanism, certainly has the potential to explain the global effects of SL signalling. However, beyond the specific examples cited here, there is currently little evidence that changes in PIN endocytosis underlie the broader effects of SL signalling. Indeed, there is no a priori reason for all SL effects to be mediated through a unified downstream mechanism. The current uncertainty surrounding downstream effects in SL signalling demonstrates that although we now have a genetically well-defined core SL signalling pathway, a priority is to develop a deeper understanding of the cell biology of this system. In the next sections, we will examine the current gaps in our knowledge, and how these may be addressed.
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Possible scheme for evolution of SL and karrikin signalling pathways. In charophyte algae, endogenous karrikin-like molecules (‘KL’, brown), and possibly SLs, signal via KAI2 proteins (light blue) and unknown effectors (red). In mosses (and other basal land plants), KAI2 signalling has become associated with SCFMAX2 (orange/purple) and leads to the degradation of SMXL proteins (light green). SLs mostly signal via the ancient MAX2-independent mechanism, but still could still plausibly interact with the newly evolved KAI2-MAX2-SMXL signalling system as well. KL might signal via both mechanisms. In angiosperms, subfunctionalization or neo-functionalization of KAI2-like and SMXL proteins, followed by co-evolution, leads to establishment of SCFMAX2dependent SL signalling via D14–D53 (dark blue/dark green). The specificity of KAI2-SMAX1 signalling may also have increased such that endogenous SLs cannot signal via KAI2 [44,45], and karrikins (KAR) and KL cannot signal via D14. It is likely that KAI2 cannot interact with D53 (and likewise that D14 cannot interact with SMAX1). However, the MAX2-binding domain of the KAI2/SMAX1 and D14/D53 pairs is not changed, such that no sub-functionalization of MAX2 occurs. MAX2independent effects of SL in Arabidopsis [2] also suggest the persistence of the ancient ‘non-canonical’ SL signalling pathway.
expression are thus only a partial explanation for the effect of SL on branching. Moreover, since BRC1 is not broadly expressed throughout the plant [31], changes in BRC1 expression seem unlikely to explain more than a small subset of the global responses to SL. In summary, while it certainly cannot be ruled out, there is little evidence that the major effects of SL signalling are mediated primarily through changes in transcription.
Protein partners for D53 The rice D53 protein contains three possible EAR motifs, which are also found in proteins that interact with TPR transcriptional co-repressors, and it has thus been suggested that D53 regulates transcription of SL response genes in a TPR-dependent manner (Figure 1b) [17,18]. Although D53 appears to bind weakly to some of the TPR proteins [17], there is currently no evidence that its EAR motifs are actually relevant for SL signalling. Moreover only one of these domains is found in SMXL6/SMXL7/ SMXL8 [18], while SMAX1 class proteins contain a divergent type of EAR motif (Table 2). An important priority for understanding SL signalling is therefore to establish the importance of the EAR motifs in D53 class proteins. Even if they are functional, EAR motifs are not exclusively associated with TPR-mediated transcriptional repression, but rather mediate interactions with proteins containing corresponding CTLH domains [37]. In Arabidopsis, several CTLH-domain containing proteins are found in a large cytoplasmic complex that regulates cytoskeletal organization [38], and in yeast CTLH-domain proteins are involved in the targeted endocytosis of fructose-1,6-bisphosphatase [39] (Figure 1b). The presence of an EAR motif in D53 proteins thus in no way predicates that they must function in transcription, and it is plausible that they function in regulating endocytosis. Either way, an important priority for understanding downstream events in SL signalling will be to identify protein partners for D53 via biochemical and genetic approaches.
Sub-cellular localization of SL signalling Removal of the auxin efflux carrier PIN1 from the plasma membrane has been suggested as a fast and direct outcome of SL signalling in Arabidopsis [33,34] (nonmutually exclusive with transcriptional responses), and has been proposed to regulate bud outgrowth via an auxin transport canalization-dependent mechanism [34,35; www.sciencedirect.com
The different distal targets proposed for SL signalling tend to imply distinct sub-cellular localizations for the SL signalling complex (Figure 1b), and thus understanding where and how SL signalling occurs within the cell may help to distinguish between these possibilities, although the current data are somewhat unhelpful in this regard. Current Opinion in Plant Biology 2014, 22:7–13
10 Cell biology
Table 1 Nomenclature and relationship of proteins described in this study. Several classes of proteins participate in SL and karrikin signalling, listed at the left; each class is named after its founding member. Members of these classes have widely varying names between species, summarized in the body of the table. When discussing SL signalling in general terms, we have used the name of the class to represent all members. When we refer to a particular species, we have used the name of the protein from that species. Protein type a/b-fold F-box
Class D14 KAI2 MAX2
Arabidopsis (At) D14 KAI2 MAX2
Rice (Os) D14 D14L D3
Pea (Ps) Petunia (Ph) DAD2 RMS4
MAX2a MAX2b
HSP101/chaperonin-like D53 SMXL6 SMXL7 MXL8 D53 SMAX1 SMAX1 [Os08g15230] TCP transcription factor TB1 BRC1 BRC2 FC1 BRC1
D14 proteins appear to be present in both the cytoplasm and nucleus [13,18], as do MAX2 proteins [18,20], while published data from protoplasts and root cells suggest a primarily nuclear localization for D53 proteins [17,18]. If SL signalling does occur in the nucleus, then it is somewhat remarkable that none of these proteins contain strong nuclear localization signals, and the relevance of cytoplasmic D14 and MAX2 is unclear. Manipulating the sub-cellular localization of SL signalling components by targeting them to specific cellular compartments could therefore be highly informative in establishing exactly where in the cell SL signalling is required for different processes. A complementary approach would be to use fluorescently labelled versions of these proteins, to analyse where in the cell they actually interact with
Function SL receptor Karrikin receptor Signalling intermediate Target of SL signalling Target of Karrikin signalling? Putative downstream target of SL signalling
each other. Bimolecular fluorescence complementation suggests that D53 and D14 from rice interact in the nucleus, but this has only been demonstrated in a heterologous tobacco system [18]. It will also be important to test the localization of each protein in genetic backgrounds mutant for the others, to assess how these proteins affect each other’s sub-cellular localization.
Cell-autonomy of SL signalling Another aspect worth examining is the expression pattern and cell autonomy of SL signalling. The published expression patterns of SL signalling components do not always match well with SL-triggered responses. For example, while MAX2 is expressed in vascular-associated cells in most organs in Arabidopsis, it does not appear to
Table 2
Rice Rice Arabidopsis Arabidopsis Arabidopsis Poplar Poplar Tomato Tomato Arabidopsis Arabidopsis Poplar Poplar Tomato Rice Physcomitrella Physcomitrella
Name/ accession number D53 D53-like SMXL6 SMXL7 SMXL8 Potri.001G252500 Potri.008G069100 Solyc09g055230 Solyc06g051460 SMAX1 SMXL2 Potri.018G097300 Potri.006G175200 Solyc07g006540 Os08g15230 Pp1s248_7V6 Pp1s30_156V6
1st position
2nd position
3rd position
LVLNLQ LVLNLQ FQGMVA YLETPK ~~~~~~ VPRLKQ CILSKQ PAQSLE SSIFLS LRQPLQ ~~~~~~ PRQPFQ PHQPFQ LRQPLQ PRPPVE ~~~~~~ ~~~~~~
LDLNLQ LDLNLQ VDFKSQ VDFGAE VDLGAA LDLGSH ADLSAQ VDLGSS VDLSLQ LGS~~~ LGS~~~ LGS~~~ LGS~~~ LGS~~~ FGGDSR LGFADG FGDLGY
FDLNLP FDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP LDLNLP FDLNQA FDLNEA FDLNEA FDLNEA FDLNEA LDLNLA LDLNLA LDLNLS
SMAX1 clade
Species
D53 clade
EAR (ethylene responsive element binding factor-associated amphiphilic repression) motifs and SMXL proteins. Three potential LxLxLtype EAR motifs have been identified in D53 from rice [17,18], but two of these are not conserved in D53 homologues from other species, suggesting they are either spurious or of limited relevance to the general function of proteins in the D53 sub-clade. The third EAR motif is well conserved across this sub-clade of the SMXL family, suggesting it is of functional relevance. EAR motifs are found in SMAX1 from Arabidopsis and other eudicot members of the SMAX1 clade, but they are of the divergent FDLN-type. The significance of this is not clear, and the rice SMAX1 homologue has an LxLxL-type motif in the same position. EAR motifs are also found in third position in SMXL homologues from the basal land plant Physcomitrella patens; these proteins do not fall into either the D53 or SMAX1 sub-clade.
Notes: Red = Leucine residues that might form part of an EAR motif, Blue = Asparagine or Aspartate residues that might form part of an EAR motif.
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Strigolactone signalling Bennett and Leyser 11
be well expressed in the root epidermis or root hair cells [20,40], both of which are sensitive to GR24 (and therefore potentially SL) [3,36]. In the case of root hair elongation, MAX2 expression in the endodermis is sufficient to control developmental effects in the epidermis, suggesting noncell autonomous outputs for SL signalling [36,41], but how this operates, and whether it is sufficient for all putative epidermal effects of SL is unclear. D14 is expressed in a broadly similar pattern to MAX2 in Arabidopsis, but distinct differences between D14 promoter and protein fusions suggest that D14 might have a degree of mobility (though it cannot act graft-transmissably) [13]. However, the consequences of this movement and whether it relates to non-cell autonomous signalling is also unclear. No detailed expression pattern for SMXL6/SMXL7/SMXL8 has been reported yet, although D53 in rice appears to be expressed in an analogous pattern to D14 and MAX2 [18]. Ultimately, more sensitive tools are required to assess exactly where SL signalling components are expressed and where they function, and whether very low expression levels are sufficient for meaningful activity. It is also likely that some of the putative non-cell autonomous effects of SLs could result from changes in auxin distribution downstream of SL signalling [36,41], and care should therefore be exercised when interpreting apparent SL responses in non-vascular-associated tissues.
Evolution and non-canonical strigolactone signalling One of the most fascinating aspects of SL signalling is its evolution, since the canonical SL and karrikin signalling pathways clearly share an evolutionary origin. The currently available evidence points to the karrikin pathway being the more ancient of the two, since the members of the D14/KAI2 clade in basal land plants and algae are unambiguously similar to KAI2 [11,42]. Members of the D14 and D53 sub-clades have so far only been detected in vascular plants [11,18], suggesting that the canonical SL signalling pathway is a relatively recent innovation, probably arising by partial duplication of the karrikin pathway, followed by sub-functionalization or neo-functionalization (and presumably co-evolution) of the new D14 and D53 pair (Figure 2). However, SLs are present in, and GR24 can regulate development in algae and basal land plants [42,43], clearly implying the existence of more ancient, non-canonical SL signalling mechanisms. One very plausible possibility is that SL signals through KAI2-SMAX1 in early land plants, due to lower specificity in the binding pocket of KAI2 (Figure 2). In Arabidopsis, endogenously occurring SLs do not seem to be able to signal via KAI2 [44,45], but it is possible that after the duplication event that created the pair, both KAI2 and D14 have evolved greater binding specificity than is present in the KAI2-like protein from which they originated (Figure 2). However, it appears that MAX2 has www.sciencedirect.com
evolved specifically in land plants [46], and so this hypothesis is insufficient to explain SL signalling in algae, which must be MAX2-independent. Furthermore, there also appear to be MAX2-independent effects of GR24 in Arabidopsis [2], and SL signalling in moss also may not act through MAX2 [47]. An intriguing hypothesis is therefore that there is a more ancient SL signalling mechanism, possibly involving a KAI2 and/or D14 family member, which has been maintained throughout land plant evolution even though largely superseded by the emergence of canonical D14-MAX2-D53-dependent signalling (Figure 2).
Conclusions The 6 year period since the identification of SLs as endogenous growth regulators has seen an explosion in interest in their biology. The establishment of a canonical signalling pathway in that short time is a remarkable achievement, and seems a good point to take stock of what we now know. Despite — or perhaps because of — the rapid progress, there remain many gaps, contradictions or uncertainties in our understanding of SL biology. What is now required is a degree of consolidation and careful analysis integrating genetic, cell and molecular biological approaches with biochemistry and structural biology to establish more firmly the fundamentals of SL signalling.
Acknowledgements Our research is funded by the Gatsby Foundation (GAT3272C) and the European Research Council (No. 294514 – EnCoDe).
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signals. This study also provides a clear framework for understanding the apparently recent evolution of canonical SL signalling. 43. Proust H, Hoffmann B, Xie X, Yoneyama K, Schaefer DG, Yoneyama K, Nogue´ F, Rameau C: Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens. Development 2011, 138:1531-1539. 44. Scaffidi A, Waters MT, Ghisalberti EL, Dixon KW, Flematti GR, Smith SM: Carlactone-independent seedling morphogenesis in Arabidopsis. Plant J 2013, 76:1-9. A thorough study demonstrating that although the SL analogue GR24 can activate KAI2-dependent signalling, endogenous SLs derived from carlactone can only signal through D14. 45. Scaffidi A, Waters M, Sun YK, Skelton BW, Dixon KW, Ghisalberti EL, Flematti G, Smith S: Strigolactone hormones and their stereoisomers signal through two related receptor proteins to induce different physiological responses in Arabidopsis. Plant Physiol 2014 May 7. A follow up to [41], examining how natural and non-naturally occurring stereoisomers of SLs act in plants. D14 displays stereoselectivity towards naturally occurring SLs, while non-naturally occurring enantiomers signal mainly through KAI2, thus showing that stereochemistry is a crucial aspect of SL signalling specificity. 46. Challis RJ, Hepworth J, Mouchel C, Waites R, Leyser O: A role for more axillary growth1 (MAX1) in evolutionary diversity in strigolactone signalling upstream of MAX2. Plant Physiol 2013, 161:1885-1902. 47. de Saint Germain A, Bonhomme S, Boyer FD, Rameau C: Novel insights into strigolactone distribution and signalling. Curr Opin Plant Biol 2013, 16:583-589.
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