Chemical genetics and strigolactone perception

Abstract

Strigolactones (SLs) are a collection of related small molecules that act as hormones in plant growth and development. Intriguingly, SLs also act as ecological communicators between plants and mycorrhizal fungi and between host plants and a collection of parasitic plant species. In the case of mycorrhizal fungi, SLs exude into the soil from host roots to attract fungal hyphae for a beneficial interaction. In the case of parasitic plants, however, root-exuded SLs cause dormant parasitic plant seeds to germinate, thereby allowing the resulting seedling to infect the host and withdraw nutrients. Because a laboratory-friendly model does not exist for parasitic plants, researchers are currently using information gleaned from model plants like Arabidopsis in combination with the chemical probes developed through chemical genetics to understand SL perception of parasitic plants. This work first shows that understanding SL signaling is useful in developing chemical probes that perturb SL perception. Second, it indicates that the chemical space available to probe SL signaling in both model and parasitic plants is sizeable. Because these parasitic pests represent a major concern for food insecurity in the developing world, there is great need for chemical approaches to uncover novel lead compounds that perturb parasitic plant infections.

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Chemical genetics and strigolactone perception [version 1;
referees: 2 approved]
ShelleyLumba, MichaelBunsick, PeterMcCourt
CellandSystemsBiology,UniversityofToronto,andtheCentrefortheAnalysisofGenomeEvolutionandFunction,UniversityofToronto,
Toronto,ON,M5S3B2,Canada
Abstract
Strigolactones(SLs)areacollectionofrelatedsmallmoleculesthatactas
hormonesinplantgrowthanddevelopment.Intriguingly,SLsalsoactas
ecologicalcommunicatorsbetweenplantsandmycorrhizalfungiandbetween
hostplantsandacollectionofparasiticplantspecies.Inthecaseofmycorrhizal
fungi,SLsexudeintothesoilfromhostrootstoattractfungalhyphaefora
beneficialinteraction.Inthecaseofparasiticplants,however,root-exudedSLs
causedormantparasiticplantseedstogerminate,therebyallowingthe
resultingseedlingtoinfectthehostandwithdrawnutrients.Becausea
laboratory-friendlymodeldoesnotexistforparasiticplants,researchersare
currentlyusinginformationgleanedfrommodelplantslike inArabidopsis
combinationwiththechemicalprobesdevelopedthroughchemicalgeneticsto
understandSLperceptionofparasiticplants.Thisworkfirstshowsthat
understandingSLsignalingisusefulindevelopingchemicalprobesthatperturb
SLperception.Second,itindicatesthatthechemicalspaceavailabletoprobe
SLsignalinginbothmodelandparasiticplantsissizeable.Becausethese
parasiticpestsrepresentamajorconcernforfoodinsecurityinthedeveloping
world,thereisgreatneedforchemicalapproachestouncovernovellead
compoundsthatperturbparasiticplantinfections.
 
Referee Status:
 InvitedReferees

version 1
published
22Jun2017

1 2
,UniversityofCalifornia,David C Nelson
USA
1
,UniversityofTasmania,Steven Smith
Australia
2
22Jun2017, (F1000FacultyRev):975(doi:First published: 6
)10.12688/f1000research.11379.1
22Jun2017, (F1000FacultyRev):975(doi:Latest published: 6
)10.12688/f1000research.11379.1
v1
Page 1 of 12
F1000Research 2017, 6(F1000 Faculty Rev):975 Last updated: 22 JUN 2017


PeterMcCourt( )Corresponding author: peter.mccourt@utoronto.ca
Competing interests: Theauthorsdeclarethattheyhavenocompetinginterests.
LumbaS,BunsickMandMcCourtP.How to cite this article: Chemical genetics and strigolactone perception [version 1; referees: 2
 2017, (F1000FacultyRev):975(doi: )approved] F1000Research 610.12688/f1000research.11379.1
©2017LumbaS .Thisisanopenaccessarticledistributedunderthetermsofthe ,whichCopyright: et al CreativeCommonsAttributionLicence
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
TheauthorswishtoacknowledgesupportfromtheNationalScience&EngineeringResearchCouncilofCanada(NSERCGrant information:
300001)toPeterMcCourtand(NSERC502592)toShelleyLumba.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
22Jun2017, (F1000FacultyRev):975(doi: )First published: 6 10.12688/f1000research.11379.1
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Introduction
Small organic molecules are important sources of signaling hor-
mones in both plants and animals1–3. With respect to plants, around
ten small molecule hormones have been identified so far, and core
signaling pathways for each of these hormones have been charac-
terized3. Much of the success in understanding how small molecule
hormones are perceived in plants comes from genetic analysis,
which usually involves finding mutants with altered hormone sensi-
tivity followed by molecular identification of the wild-type protein
involved. The power of genetics in dissecting plant hormone sign-
aling was impressive, especially since many players in plant hor-
mone signaling were genetically redundant, which precluded their
identification as simple recessive mutations4–6. Many saturated
genetic screens, however, led to the identification of rare dominant
mutations7–9 which served as toeholds in building many signal-
ing pathways. The subsequent development of well-characterized
mutant knockout collections in Arabidopsis10 allowed more com-
ponents to be validated through the construction of multiple loss-
of-function mutant lines in redundant steps.
The contribution of genetics to unravelling plant hormone biol-
ogy is self-evident, which raises interest in where genetic analysis
can make future contributions. A good example of the evolution of
genetic approaches in plant hormone signaling is now occurring in
the field of chemical genetics11. Simply defined, chemical genet-
ics involves the development of chemical agonists and antagonists
to probe biological processes. This approach by definition should
be well suited for plant hormone signaling since plant hormones
are small molecules and as such their receptors should be “drug-
gable”12. However, geneticists often look suspiciously on chemical
perturbation experiments because of concerns of off-target effects.
Chemical geneticists have tried to address this criticism by defining
criteria for what makes a good chemical probe13,14 (Table 1). From a
sceptic’s perspective, two of these conditions go far to assuage their
chemical fears. First, chemical addition to wild-type organisms
must clearly and specifically mimic a well-characterized mutant
phenotype. For example, the addition of silver ions or 1-methylcy-
clopropene, both of which antagonize ethylene receptors, results in
dark-grown seedlings that look phenotypically similar to mutations
that decrease ethylene perception15. A second and more powerful
criterion that monitors off-target effects is the use of a “decoder
strategy”16. In this case, application of the compound in question
to a loss-of-function mutant in the target gene should result in loss
of the global gene expression signature generated by compound
treatment of the wild-type. For example, addition of the histidine
biosynthetic inhibitor 5-amino triazole (5-AT) to a mutant lacking
the 5-AT target (histidine3 [his3]) resulted in dramatic reductions
in the global gene expression signature observed in 5-AT-treated
wild-type yeast cells16.
In plants, perhaps the best example of chemical probe development
involved the identification of pyrabactin as a selective agonist of
the receptor for the hormone abscisic acid (ABA)17. Pyrabactin
was first identified as a general germination inhibitor in a chemical
screen in Arabidopsis, but its specific role in ABA signaling was
suggested by the ability of ABA-insensitive mutants to germinate
on pyrabactin. Second, the pyrabactin analog, apyrabactin, showed
no biological activity. Finally, although a decoder approach was not
applied, global gene expression experiments between seeds treated
with ABA or pyrabactin were highly correlated, suggesting few off-
target effects.
The identification of pyrabactin allowed the development of genetic
screens to identify mutations in an essential gene encoding an ABA
receptor that is involved in germination17. Because pyrabactin was
a selective ABA agonist, it activated only a subset of ABA recep-
tors and particularly the major one involved in germination17. Thus,
resistant mutants to pyrabactin circumvent genetic redundancy
issues that cannot be resolved by traditional ABA screens. This
result showed how a new approach like chemical genetics, which
can identify more selective compounds, can uncover novelty when
wedded to an old approach like a traditional forward genetics
screen. The pyrabactin story also demonstrated how the identifi-
cation of a specific chemical probe can have broader applications
beyond basic biology. Information on the pyrabactin structure has
led to the identification of chemical analogs that could be used to
protect important agronomic crops from drought, a process that is
mediated by ABA signaling18,19.
This development of chemicals as both probes for plant hormone
signaling and translational leads is clearly exciting. Perhaps there
is not a more obvious application of chemicals than in the study
of a recently identified collection of chemically related hormones
called strigolactones (SLs)20,21. SLs, like all small molecule plant
hormones, have many roles in plant growth ranging from filament
growth in nonvascular plants22 to shoot branching23 and root devel-
opment24 in vascular plants. However, unlike most plant hormones,
SLs also have ecological signaling roles. SLs are exuded from plant
roots into the rhizosphere, where they attract arbuscular mycor-
rhizal (AM) fungi for a beneficial interaction that brings water and
nutrients to the plant25. Unfortunately, root-exuded SLs also act as a
cue to tell a number of parasitic plant species that a host is nearby26.
In these cases, obligate parasites of the Striga, Phelipanche, and
Orobanche genera have evolved strong seed dormancy, which is
broken only when they sense host-derived SLs. After germination,
the parasite infects the host with devastating consequences. The
Table 1. Some criteria used for determining the utility of a
chemical probe.
Chemistry
Structure Defined structure
Stability Stable in test media
Potency
Biochemical <100 nM in in vitro biochemical assay
Cellular <1–10 mM in cell or whole organism assay
Analogs Closely related structures have similar activity
Selectivity
Inactive
analogs
Analogs with no biochemical activity have no
biological activity
Genetic Chemical closely mimics mutant phenotypes
Target Chemical follows “decoder” parameters16
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range of infestations by the Striga genera alone includes much of
sub-Saharan Africa, and these infestations are considered to be a
major impediment to poverty alleviation on the continent27,28. Thus,
the identification of chemicals that probe SL perception not only
will help to understand SL biology but also may lead to the devel-
opment of new compounds to combat parasitic plant infestations in
the developing world.
SL chemistry and signaling
The application of chemical genetics to understanding and perturb-
ing SL functions in parasitic plants requires a basic understanding
of both SL chemistry and signaling. The canonical structure of a SL
molecule is usually represented as a butenolide ring (D ring) con-
nected to a tricyclic lactone (ABC rings) via an enol-ether bridge
(Figure 1)29,30. The ABC rings do show natural chemical variation,
and to date approximately 20 SLs have now been identified29,30. SLs
can be further categorized based on stereochemistry around the
B and C ring into the strigol and orobanchol families (Figure 1).
Finally, stereochemistry at the C2’ position appears to be important,
with the natural R-isomer showing the best SL activity31–34.
Further hints as to what parts of a SL molecule are important for
perception have come from both natural and synthetic sources35.
Avenaol, for example, which is found in root exudates from Avena
strigosa (black oat) and shows SL activity, has the C and D moie-
ties but lacks a B ring and has an additional carbon between the A
and C rings (Figure 2)36. Synthetic compounds, such as GR7 and
GR5, that lack A and B rings but retain CD ring chemistry also
show activity in both parasitic and nonparasitic plants, whilst com-
pounds that contain the ABC rings but lack a D ring are inactive
(Figure 2)33–35. Together, these chemical variants suggest that the
C and D rings are essential for SL activity.
Figure 1. Chemical structure of strigolactone (SL). The chemical structures of naturally occurring SLs can be divided into two families, the
orobancol family (a) and the strigol family (b) based on stereochemistry around the BC ring. Chemical differences within a family are related
to substitutions (R) on the A or C rings. All naturally occurring SLs found to date have C2’-(R) stereochemistry via the enol-ether bridge that
connects the C and D rings. GR24 (c) shown in the C2’-(R) conformation is the most commonly used synthetic SL.
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The enol-ether linkage between the C and D rings also seems to
be important. SL receptors in model plants, collectively called
D14-type SL receptors after the rice DWARF14 (D14) receptor37,
metabolize SLs to generate tricyclic ABC and D ring
moieties38. Structurally, D14-type receptors have a canonical
α/β-fold consisting of four β-sheets bound by a collection of four
α-helices (αT1–αT4), which form a lid encompassing the SL lig-
and-binding pocket (Figure 3)38–42. Within the pocket, there is a
functional serine–histidine–aspartate catalytic triad that is required
for SL hydrolysis38,41. Although the order of signaling events after
an SL binds a D14-type receptor still needs to be clarified, one
simple path appears to be emerging (Figure 3). As SL enters the
ligand-binding pocket, a catalytic serine (Ser96 in the D14 pro-
tein from rice) attacks the C5’ position of the D ring, resulting in
cleavage and release of the tricyclic ABC ring43,44. The remaining
D-ring moiety covalently bonds with the catalytic histidine (His247).
This event appears to result in small conformational changes that
allow the recruitment of other SL signaling partners. The recruit-
ment of these signaling partners in turn appears to cause a larger
conformational change of the receptor to a closed state by trapping
either a D-ring intermediate or the D ring itself43. Although many
details on timing and order need to be worked out, the covalent
bond between the D-ring moiety and D14-type receptors suggests
that the metabolism of the SL ligand may be integral to perception
and downstream SL signaling44. This also explains why D14-type
receptors retain a canonical α/β hydrolase amino acid catalytic triad
that shows very slow kinetics of hydrolysis38,41,44.
So what about parasitic plant SL signaling? Unlike model plants,
D14-type receptors do not appear to be the major player in the ger-
mination response of parasites to host-derived SLs. This function
falls to a related D14 α/β hydrolase given the name HYPOSEN-
SITIVE TO LIGHT/KARRIKIN/INSENSITIVE2 (HTL/KAI2)45.
The double-barreled name of HTL/KAI2 is because of two groups
independently identifying loss-of-function mutations in this gene
based on two phenotypes: 1) hyposensitivity to light (HTL)46 and 2)
insensitivity to the smoke-derived germination stimulant karrikin
(KAI2)47. The identification of HTL/KAI2 hydrolases as SL recep-
tors in parasitic plants was surprising, as these proteins in model
systems do not respond well to naturally occurring C2’-(R) SL iso-
mers and at this time their natural ligand is unknown48. Analysis of
HTL/KAI2 genes from Striga hermonthica (ShHTL/KAI2) revealed
that these receptors have a range of sensitivity and specificity with
respect to the SLs they recognize49,50. Functional analysis in Ara-
bidopsis demonstrated that one receptor, ShHTL7, was sufficient
to increase the sensitivity of the SL response in Arabidopsis to
picomolar levels, which is within the concentration range observed
for the response of Striga hermonthica seed to SLs50. Recent bio-
chemical analysis of ShHTL7 suggested that it may have a similar
mode of action to D14-type receptors with respect to transducing
an SL signal51. Although a mechanistic understanding of how para-
sitic plant receptors attain high levels of SL sensitivity has not been
clearly elucidated, it does appear that parasitic plant HTL/KAI2
genes, unlike their nonparasitic plant counterparts, have evolved
away from perceiving karrikins to sensing SLs45,50.
Figure 2. Chemical structures of some strigolactone (SL) agonists with butenolide rings. (a) Each of these SL agonists has an attached
butenolide and is expected to be hydrolyzed by D14-type hydrolases. (b) These debranones are three monohalogenated derivatives. The
chlorinated derivative on the right is moderately active in Striga hermonthica germination assays. (c) Yoshimulactone green (YLG) is hydrolyzed
by SL receptors to yield a D ring and fluorescein that fluoresces green.
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Chemical screening for SL agonists
The requirement of host-derived SLs for the germination of parasitic
plant seed has been a major focus on which to develop strategies to
combat these pests52,53. The logic involves synthesizing cheap SL
analogs that are stable in the soil which would germinate parasitic
seed in the absence of a host. These compounds are generically
called “suicide germination compounds” because these obligate
parasites like Striga hermonthica will die after germination without
a host. The first synthesized suicide germination compounds such
as the popular derivative GR24 (Figure 1) were designed around the
SL core structure, but it soon became clear that the chemical space
for SL activity was not limited to canonical SL scaffolds. Partially
this occurred as hormone-based SL bioassays became routine35. For
example, rice54 and pea34 researchers used rescue of SL-deficient
branching phenotypes as a screening tool to determine SL activity of
their compounds. This approach led to the identification of phenoxy
furanone derivatives called debranones (de-branching furanones)
that showed good SL activity although they lacked the ABC ring
structures and any enol-ether linkages (Figure 2). This observation
that enol-ether chemistry was not required for activity was in part
responsible in the development of on-off fluorescent probes such as
yoshimulactone green (YLG) that were instrumental in understand-
ing the SL responses in parasitic plants like Striga hermonthica
(Figure 2)49. Interestingly, although YLG acts as a SL in both para-
sitic plant germination and nonparasitic plant branching, debranone
was not potent in parasitic plant germination assays54,55. However,
specific chlorine additions on the phenyl ring of the debranone scaf-
fold (Figure 2) do improve the response of parasitic seeds55. This
implies that compounds can be developed that preferentially target
the pests without influencing host behavior.
This idea of using biology rather than chemistry to guide SL activ-
ity has greatly expanded as good large annotated chemical libraries
became commercially available11. For example, a chemical screen
designed strictly around Arabidopsis germination and early seed-
ling growth identified a collection of succinimide and phthalimide
compounds that appeared to impinge on SL biology56. Collectively
called cotylimides (CTLs), a number of these compounds have
subsequently been shown to bind and activate AtHTL/KAI257. The
phthalimide structure in some cotylimides (CTL-IV) is also found
in Nijmegen-1, a potent SL mimic58. However, unlike Nijmegen-1,
CTL compounds do not contain a D ring and certainly are not a
hydrolysable substrate. Interestingly, other compounds built on
phthalimide lactone scaffolds but lacking enol-ether linkages have
good selectivity activity on various Orobanche and Philibanche
species59. Finally, a screen for novel germination agonists devel-
oped specifically around the activation of the AtHTL/KAI2 receptor
with one of its protein partners, MORE AXILLARY GROWTH2
(MAX2), identified more structurally unrelated compounds that
have activity in Striga hermonthica germination assays (Figure 4)57.
Figure 3. A model of strigolactone (SL) perception. A D14-type receptor without SL is in an unbound open conformation (open). Upon
SL binding, the SL is hydrolyzed, releasing the ABC rings. Hydrolysis occurs via a nucleophilic attack by the S96 amino acid of the catalytic
triad, releasing an ABC ring. A covalent bond then occurs between the C5’ moiety of the D ring and H247, leading to a D-ring intermediate.
During this process, it is thought that D14 signaling partners are recruited, which promotes transition of the receptor to a closed state (closed).
Receptor transition from an open to a closed configuration is represented by models of four intermediate crystal structures (pink, within the
brackets). Below each crystal is a tube representation of the four α helices (αT1–αT4) that form a lid and their positions during the transition
from an open to a closed state. As the receptor transitions, the αT1 (brown), αT2 (green), and αT4 (yellow) helices move to close the lid. The
αT2 helix becomes an unordered ribbon (blue). A movie of the transition from the open to the closed form can be found in Supplementary File 1.
The exact order of events after SL hydrolysis with respect to conformational changes and recruitment of signaling partners remains to be
clarified. Based on in vitro analysis, it is unclear whether the D ring is irreversibly trapped within the closed receptor. The open structure has
the PDB code 4IH4. The closed structure has the PDB code 5HZG.
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Figure 4. Chemical structures of some strigolactone (SL) agonists lacking butenolide rings. (a) Nijmegen-1 (upper left corner), which
contains a butenolide ring, is shown as a reference phthalimide-based SL mimic. The phthalimide lactone core structure (lower left corner)
was used to develop new parasitic plant germination stimulants. R-groups represent different places on the core structure where modifications
were made. (b) The structures of these three compounds were found by screening for compounds that encourage protein–protein interactions
between AtHTL/KAI2 and its F-box partner protein MAX238.
Thus, biologically based screens appear to be uncovering a myr-
iad of chemical compounds that show little chemical relatedness
to canonical SLs. The diversity of compounds with SL activity is
surprising, as early work suggested that CD rings and stereochem-
istry are vital to the SL response. Furthermore, all the known SL
receptors have a conserved catalytic triad, indicating that hydrolysis
is essential to signaling. Possibly, these new SL agonists bind SL
receptors in different places than the canonical SLs and as such
do not require hydrolysis for activity. Alternatively, it would also
be interesting to test these compounds on catalytically dead tirad
mutants. Whatever the case, structural studies with these different
compounds will be needed to resolve these issues.
Chemical screening for SL antagonists
Although chemical genetic screens for new SL agonists are viewed
through the prism of SL chemistry, screening for SL antagonists has
not had this bias. One of the first attempts to identify SL antago-
nists involved taking advantage of D14-type receptor structural
biology. Compounds were first screened in silico for chemicals that
would theoretically fit the binding pocket of the rice D14 receptor60.
Positive hits were next tested using a yeast two-hybrid assay to
find which compounds reduced SL-dependent D14-protein-protein
interactions. This approach identified 2-methoxy-1-naphthalde-
hyde (2-MN) (Figure 5), and subsequent experiments showed
2-MN interfered with a number of SL-dependent processes in
rice and Arabidopsis60. 2-MN showed some activity in inhib-
iting Striga hermonthica seed germination, and this reduced
potency may reflect the experimental design that was based
on D14-type receptors rather than parasitic plant HTL/KAI2
receptors. A crystal structure of the SL receptor (ShHTL5) from
Striga hermonthica now exists50, and it would be interesting to
see how in silico screening of this receptor may influence lead
compound identification.
Recently, a second SL antagonist screen has been performed
using whole plant SL-dependent assays in Arabidopsis rather than
knowledge of the receptor structure a priori. This assay was based
on the observation that SLs inhibited Arabidopsis hypocotyl
elongation. Another advantage to using Arabidopsis was a geno-
type whose hypocotyl was sensitized to SL inhibition, which
was used in the primary screen to identify compounds that inter-
fered with SL-dependent inhibition of hypocotyl growth61. Seven
compounds were found, one of which was a piperidine-based
molecule called soporidine (SOP). In addition to binding AtHTL/
KAI2, SOP bound the Striga hermonthica receptor ShHTL7 and
inhibited its hydrolytic activity. Functionally, SOP inhibited the
germination of Striga hermonthica seed in the presence of SL61.
Thus, hypocotyl-based assays in Arabidopsis appear to be a good
screening platform to identify lead compounds that may per-
turb Striga germination. None of the antagonists identified so far
have significant structural similarity to known naturally occurring
SLs. Similar to the case of SL agonists, it is possible that these
antagonists bind somewhere else on the receptor to inhibit activity.
This again begs for more studies on the structure–function rela-
tionships between the SL receptors and the plethora of compounds
identified from chemical screens.
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Figure 5. Chemical structures of some strigolactone (SL) antagonists. Within these antagonists, only 2-methoxy-1-naphthaldehyde
(2-MN) and soporidine (SOP) have been closely characterized biochemically in model systems and with respect to Striga hermonthica
germination.
Concluding remarks
The rapid increase in our knowledge of the fundamental biology
of SL signaling based on studies on model plants is allowing
researchers to test the mechanism by which SLs are perceived in
parasitic plants. Furthermore, this information is currently being
used to identify new chemical probes that perturb SL percep-
tion in both nonparasitic and parasitic plants. Although it is not
surprising that small molecules resembling SLs have activity in
SL-based plant assays, the identification of a large number of syn-
thetic lead compounds from chemical screens that do not possess
canonical SL structures is intriguing. This could simply reflect dif-
ferent binding sites and modes of action. It could also mean that
SL receptors, whether from parasitic or nonparasitic plants, are
more promiscuous than previously thought with respect to small
molecule activation, particularly at higher concentrations.
Biologists are usually taught that compounds that bind a receptor
with high affinity are most likely the most relevant in vivo. This
explanation, however, has never really explained functional low-
affinity ligands in vivo and many times these compounds are
written off as nonspecific and biologically irrelevant. In some sense,
this hand-waving argument is akin to early beliefs about “sticky
proteins” found in large-scale protein–protein interaction networks.
Once thought to be biochemical artifacts, most of these proteins
are now viewed as essential components in scale-free signaling
networks62,63. It is now becoming clear from evolutionary studies on
small molecule hormone receptors from animals that many small
molecule receptors were initially low-affinity sensors for a range
of metabolites and later evolved to become high-affinity receptors
of particular chemicals64–66. These models are consistent with
suggestions that the ancient HTL/KAI2 receptors may have had
reduced chemical specificity that later evolved into high-specificity
SL receptors67. Such a model may explain why HTL/KAI2-type
receptors were selected over D14-type receptors by parasitic plants,
since these species must be able to readily evolve to new host SL
ligands and compositions in order to move to new hosts68.
The lack of high-affinity agonists and antagonists could suggest
chemical genetics will not yield useful probes and interesting
insights. However, although traditionally searches for drugs have
been based on finding high-affinity compounds, the pharmaceuti-
cal industry has changed its strategy to perform “fragment-based
screening”, which is designed to find low-molecular-weight com-
pounds that work in the high micromolar range initially69. Once
compounds have been identified, the methods of medicinal chem-
istry can be used to increase their potency orders of magnitude.
In this scenario, lead compounds identified with SL activity would
serve as chemical scaffolds for the development of more potent
compounds. Applying the same logic, perhaps the addition of
D-ring structures to leads implicated in SL function, would increase
their efficacy.
Finally, insights into plant versus animal hormone signaling have
come from different approaches. Animal cell culture systems2,
which are well defined developmentally and easy to handle experi-
mentally, were instrumental in dissecting how small molecule
hormones are perceived and signal. By contrast, plant hormone sign-
aling was led by phenotypic screening typically on whole organisms
Page 8 of 12
F1000Research 2017, 6(F1000 Faculty Rev):975 Last updated: 22 JUN 2017

such as Arabidopsis70. Chemical genetic analysis of hormone sign-
aling in these two kingdoms will most likely follow the same route,
and animal researchers are now more frequently using phenotypic
screening as an effective method of drug discovery71. In plant sys-
tems, compounds will continue to be identified through some phe-
notypic screen and the resulting compounds will be the basis for
mutational analysis to further understand the mode of action as was
seen with pyrabactin17. The grounding of plant chemical genetics in
phenotypic screening bodes well for new insights, since phenotypic
screening is gathering momentum as a way to re-energize animal
drug research71,72. In this sense, plant biologists are already there.
Competing interests
The authors declare that they have no competing interests.
Grant information
The authors wish to acknowledge support from the National Sci-
ence & Engineering Research Council of Canada (NSERC 300001)
to Peter McCourt and (NSERC 502592) to Shelley Lumba.
The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Supplementary material
Supplementary File 1: Changes in SL receptor structure upon SL binding. A movie of the dynamic change in the Arabidopsis D14
(AtD14) SL receptor conformation upon binding of SL as shown in Figure 2. In its open form (Open) the receptor has an accessible binding
pocket to accommodate an SL molecule. Upon binding the receptor begins conformational change that attracts singling partners result-
ing in a closed form (Closed) that traps a D-ring intermediate within the receptor (see Figure 2). In this movie only the receptor is shown
(purple).
Click here to access the data.
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
Open Peer Review
Current Referee Status:
Editorial Note on the Review Process
arecommissionedfrommembersoftheprestigious andareeditedasaF1000FacultyReviews F1000Faculty
servicetoreaders.Inordertomakethesereviewsascomprehensiveandaccessibleaspossible,thereferees
provideinputbeforepublicationandonlythefinal,revisedversionispublished.Therefereeswhoapprovedthe
finalversionarelistedwiththeirnamesandaffiliationsbutwithouttheirreportsonearlierversions(anycomments
willalreadyhavebeenaddressedinthepublishedversion).
The referees who approved this article are:
Version 1
SchoolofBiologicalSciences,UniversityofTasmania,Hobart,TAS,7001,AustraliaSteven Smith
Nocompetinginterestsweredisclosed.Competing Interests:
1
DepartmentofBotanyandPlantSciences,UniversityofCalifornia,Riverside,CA,92521,David C Nelson
USA
Nocompetinginterestsweredisclosed.Competing Interests:
1
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