Planta (2016) 243:1311–1326

DOI 10.1007/s00425-015-2455-5

REVIEW

Strigolactones: new plant hormones in action

Binne Zwanenburg1,2 • Tomáš Pospı́šil2 • Sanja Ćavar Zeljković3

Received: 4 November 2015 / Accepted: 18 December 2015 / Published online: 2 February 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract phytohormones in various biological actions, the design of

Main conclusion The key step in the mode of action of SL analogs and their applications.
strigolactones is the enzymatic detachment of the
D-ring. The thus formed hydroxy butenolide induces Keywords Karrikins  Mode of action  Signal
conformational changes of the receptor pocket which transduction  Strigolactones  Strigolactone analogs 
trigger a cascade of reactions in the signal transduction. Strigolactone mimics
Abstract Strigolactones (SLs) constitute a new class of
plant hormones which are of increasing importance in plant Abbreviations
science. For the last 60 years, they have been known as AM fungi Arbuscular mycorrhizal fungi
germination stimulants for parasitic plants. Recently, sev- KAR Karrikins
eral new bio-properties of SLs have been discovered such SL Strigolactone
as the branching factor for arbuscular mycorrhizal fungi,
regulation of plant architecture (inhibition of bud out-
growth and of shoot branching) and the response to abiotic
factors, etc. To broaden horizons and encourage new ideas
for identifying and synthesising new and structurally sim- Introduction
ple SLs, this review is focused on molecular aspects of this
new class of plant hormones. Special attention has been Strigolactones (SLs) constitute a new class of plant hor-
given to structural features, the mode of action of these mones which are of increasing importance in plant science.
They belong to the group of biologically active molecules
called semiochemicals that are used to disseminate infor-
A contribution to the special issue on Strigolactones. mation between individual species. Important examples of
plants that have become completely dependent on allelo-
& Binne Zwanenburg chemicals are the parasitic weeds witchweed (Striga spp.,
b.zwanenburg@science.ru.nl
Orobanchaceae/Scrophulariaceae) and broomrape (Oro-
1
Department of Organic Chemistry, Institute for Molecules banche spp., Orobanchaceae). The seeds of these weeds
and Materials, Radboud University Nijmegen, only germinate in response to specific chemicals, namely
Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands germination stimulants, present in the rhizosphere of host
2
Department of Growth Regulators, Faculty of Science, plants and some non-host plants. For these parasitic flow-
Centre of Region Haná for Biotechnological and Agricultural ering plants, which are totally dependent on specific
Research, Palacky University, Slechtitelu 27,
association with a host that provides nutrients and water,
78371 Olomouc, Czech Republic
3
this system ensures that germination only starts when
Central Laboratories and Research Support, Faculty of
suitable host roots are available in the immediate vicinity.
Science, Centre of Region Haná for Biotechnological and
Agricultural Research, Palacky University, Slechtitelu 27, Other allelochemicals are required to effect attachment of
78371 Olomouc, Czech Republic the germinated seeds to the roots of the host plants via a

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specialised organ, the haustorium. Once the vascular con- Isolation of SLs
nections between host and parasite have been established,
the parasite can develop at the expense of the host plant. As The first SL ever isolated was obtained from root exu-
a consequence of providing nutrients to the parasite, the dates of cotton (Gossypium hirsutum L.) as early as 1966
crop yield of the host plant will be severely affected. In and was named strigol (Cook et al. 1966). The gross
many cases of important food crops, this parasitic inter- structure of strigol was elucidated in 1972 (Cook et al.
action causes a serious problem in food production. 1972) and the full details were determined by means of an
In recent years several new bio-properties of SLs have X-ray diffraction analysis in 1985 (Brooks et al. 1985)
been discovered. A real breakthrough was the discovery about 20 years after its isolation. Strigol was isolated
that SLs act as the branching factor for arbuscular myc- from a non-host for the parasitic weed Striga and con-
orrhizal (AM) fungi (Akiyama et al. 2005; Parniske 2008). sequently, its significance for the host–parasite interaction
Mycorrhizae are symbiotic associations between soil, fungi was uncertain for a long time. It was not until 1992 that
and plant roots (Akiyama and Hayashi 2006). This inter- sorgolactone, a compound with a structure similar to
action is probably the most widespread and significant strigol, was isolated (Hauck et al. 1992) from root exu-
symbiosis in nature (Brachmann and Parniske 2006). AM dates of a true host for Striga, sorghum (Sorghum bicolor
fungi are obligate symbionts unable to complete their life L. Moench).
cycle in the absence of a suitable host. A critical step in the Soon thereafter, alectrol was obtained from the root
development of AM fungi is the triggering of the hyphal exudate of cowpea (Vigna unguiculata L.) which is a
morphogenesis by a branching factor. The isolation and host for S. gesnerioides (Muller et al. 1992). The col-
characterization of a branching factor was extremely dif- lective name ‘strigolactones’ was proposed by Butler, a
ficult due to the fact that their concentrations were very pioneer in this area (Butler 1995). The isolation of SLs
low. The first branching factor was isolated from the roots from root exudates is very laborious and requires a
of hydroponically grown Lotus japonica and it was shown careful chromatographic separation accompanied by
to be (?)-5-deoxystrigol. It was also demonstrated that bioassays for germination of appropriate seeds of para-
other SLs, such as strigol and orobanchol, are highly active sitic weeds. The production of SLs per plant is very
branching factors. Knowing the identity of the branching small: 15 pg/day/plant (Sato et al. 2005), hence collection
factors of AM fungi opens new windows for their practical of root exudate from hydroponically grown host plants
applications (Akiyama and Hayashi 2006). requires an experimental set-up with many plants. At
A second important breakthrough in SL research fol- present the HPLC separation techniques are much more
lowed a few years later. It was then demonstrated that sophisticated and fewer plants are needed. The structural
endogenous SLs play an important role in the control of analysis of SLs is a highly demanding exercise using
plant architecture. Inhibition of bud outgrowth and inhi- high resolution mass spectrometry and NMR analysis.
bition of shoot branching are typical examples (Gomez- Especially, establishing the correct stereochemistry needs
Roldan et al. 2008; Umehara et al. 2008). The inhibitory utmost care.
processes are regulated by endogenous cues of which SLs SLs invariably contain three annelated rings, the ABC
are probably most prominent. Importantly, inhibition of scaffold, connected by means of an enol ether unit with a
shoot branching could also be induced exogenously by butenolide ring, the D-ring (Fig. 1).
treatment with the synthetic SL GR24. For a long time,
involvement of two other classes of plant hormones,
namely auxin and cytokinines, has been known in con- Naturally occurring SLs
trolling shoot branching. Now, SLs are recognised as a
third class of new plant hormones. This control of plant At present two families of naturally occurring SLs are
architecture with SLs gave rise to an avalanche of publi- known (Fig. 1). Because of the tricky aspects of the
cations on this topic, indicating the high importance of this structural analyses, some misassignments were made.
new role of SLs. Several excellent reviews have appeared For example, establishing the structure of alectrol
on this subject (Tsuchiya and McCourt 2009; Koltai 2011, (Muller et al. 1992) was particularly difficult and it took
2014, 2015; Cheng et al. 2013; Waldie et al. 2014). about two decades before the correct structure was
In this review, the focus will primarily be on molecular elucidated (Ueno et al. 2011, 2015). The structure of
aspects of this intriguing class of new plant hormones. The orobanchol, which is probably one of the most abundant
synthesis of SLs, of both naturally occurring SLs and of SLs, was initially incorrectly assigned (Ueno et al.
synthetic analogs, are reviewed separately (Zwanenburg 2011). Originally it was a logical assumption that the
et al. 2015). stereochemistry would be as in (?)-strigol (Mori et al.

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Fig. 1 Structures of naturally

occurring SLs. Names of the
orobanchol family using (?)-
strigol as the parent compound
are given within brackets

1999). A third example is solanacol. In the first pro- Relevance of stereochemistry in SLs
posed structure the methyl substituents in the A-ring
were positioned para (Xie et al. 2007) instead of ortho Establishing the stereochemistry at the respective stere-
(Takikawa et al. 2009), and as far as the stereochemistry ogenic centers was, and still is, a major obstacle in eluci-
is concerned: it belongs to the orobanchol family and dating the correct detailed structure of naturally occurring
not to the strigol family as suggested originally (Chen SLs. For assigning the stereochemistry at C-20 of the D-ring
et al. 2010, 2013). More details about the structural the empirical rule reported by Welzel et al. (1999), based
corrections have been reviewed earlier (Zwanenburg and on the Cotton effect in ORD/CD spectra, is appropriate.
Pospisil 2013). For the ABC part, correlation diagrams with compounds of
The occurrence of SLs in nature and the source from known stereochemistry are mostly used (Zwanenburg and
where they have been obtained has recently been reviewed Pospisil 2013). An X-ray diffraction analysis is the most
and where possible correct structures are included in the reliable manner to establish the absolute stereochemistry of
tables (Cavar et al. 2015). Moreover, strigolactones play a an SL. However, for that a crystalline sample of the SL is
major role in host specificity of Orobanche and Pheli- needed which is not always easy to obtain. The stereo-
panche (the broomrapes) seed germination. In general, chemistry has a pronounced effect on the germinating
weedy broomrape species are less specialised in germina- activity towards the seed of parasitic weeds. In addition, for
tion requirements than the non-weedy species (Fernandez- the other SL bio-properties there is a profound effect of the
Aparicio et al. 2011). stereochemistry on the bio-response.

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Naming protocol for SLs biological processes, model compounds were designed and
prepared. A prerequisite is that these SL analogs have a
The SLs have several chiral centers, for example strigol has (much) simpler structure than natural SLs, but that their bio-
three such centers and there are 23 = 8 conceivable activity is largely retained. For a rational design of SL ana-
stereoisomers. From a chemical point of view a correct and logs, it is necessary to identify the bioactiphore, i.e. that part
unambiguous manner to designate the chirality at the of the molecule that is primarily responsible for bioactivity.
respective stereogenic centers, the use of the Cahn-Ingold- To this end the structure of a natural SL, say strigol, is sys-
Prelog (CIP) descriptors R and S to indicate the sense of tematically simplified. Making the A-ring aromatic leads to a
chirality is most appropriate. The R,S notation is based on compound which is code named GR24 after its inventor
abstract rules which are not easy to handle. Using the ent and Gerald Rosebery, removal of the A-ring gives GR7 and
epi prefixes is much easier in practise, whereby ent refers to cutting of the B-ring leads to GR5. All these GR compounds
enantiomer, i.e. mirror image of an entire unit and epi refers are appreciably active as germination stimulant for parasitic
to epimer, i.e. opposite configuration at a given atom. For the weeds (Fig. 2). However, when the C-ring is removed the
ent/epi method it is necessary to choose a reference com- activity is lost. This implies that the bioactiphore resides in
pound, a parent molecule. In the time before the structural the CD part of SLs. The information presented above allows
correction of orobanchol, the naming of SLs was simple and the design of a model compound for SL analogs with ger-
straightforward: (?)-strigol was the logical parent com- minating activity (Fig. 3). A typical feature of the model is
pound and the stereochemistry of all other SLs was related to that there is a considerable molecular freedom in the A-ring
that parent compound. However, after the structure change part of the molecule. Stereochemistry is important as men-
of orobanchol in 2011 (Ueno et al. 2011) there were two tioned in the preceding section. This model has been used
options, either to keep the naming protocol with (?)- strigol successfully to design a large series of highly active SL
as the parent or to use the new structure for natural oroban- analogs. Some typical examples are shown in Fig. 4.
chol as parent compound for the orobanchol family. Both It is important to note that these analogs not only must
methods are in use, which may lead to confusing situations have a simplified structure with retention of germinating
(Zwanenburg and Pospisil 2013). The reader is forewarned. activity, but also they must be synthetically readily
Scaffidi et al. (2014) suggested an alternative naming accessible. An illustrative example of the successful
and notation in the structural correlation of GR24 implementation of the model is Nijmegen-1. It can indeed
stereoisomers using both (?)-strigol and (-)-orobanchol as readily be obtained from simple starting materials in a few
standards. This resulted in two names for some stereoiso- synthetic steps and its germinating activity is comparable
mers, e.g. ent-20 -epi-5-deoxystrigol is also named to that of GR24.
4-deoxyorobanchol. This method has little added value and An alternative way of designing SL analogs with ger-
is confusing for those who are less familiar with stereo- mination capabilities is isosteric replacement of a particular
chemical issues. atom; in the case of SLs, most logically an oxygen atom is
replaced by another heteroatom. There are two successful
examples of such an isosteric replacement, namely: imino
Simplified SLs with retention of germinating SL analogs (Kondo et al. 2007) and strigolactams (Lachia
activity: design of SL analogs et al. 2015) (Fig. 5). In the imino analogs the electron-
withdrawing CN is essential for activity.
Naturally occurring SLs have a too complex structure for GR24 is commonly used as standard in germination
synthesis on a multi-gramme scale (Zwanenburg et al. 2015). studies. Mostly, this stimulant is a racemate in which the
The total synthesis of several natural SLs has been accom- relative stereo configuration is as in (?)-strigol. However,
plished, but linear sequences of many steps, [20 or more, it should be noted that not all seeds of parasitic weeds do
were required. To study the effect of SLs on various respond to GR24, for example O. crenata, O. foetida, O.

Fig. 2 Simplification of SL structures (all are appreciably active as germinating agents)

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SL mimics, as they mimic the SL activity but do not

have the typical SL structural features: a D-ring con-
nected with an a,b-unsaturated carbonyl via an enol
ether unit. One group of substituted D-ring compounds
with germinating activity has an aryloxy substituent at
C-5 (Fig. 6). These compounds were named as debra-
nones (branching furanones) because the main activity
profile is inhibition of shoot branching (Fukui et al.
2011, 2013). Seeds of Striga hermonthica respond
modestly to these debranones. It was found that para-
chlorophenoxy-debranone had the highest activity. So far
Fig. 3 Model for designing SL analogs with germinating activity Orobanche seeds were not tested with debranones. The
second group of SL mimics that was discovered almost
hederae and O. densiflora (Fernandez-Aparicio et al. at the same time has an aroyloxy substituent at C-5 of
2011), as well as O. picridis and O. minor subsp. maritima the D-ring (Zwanenburg et al. 2011, 2013). These SL
(Thorogood et al. 2009) do not respond. mimics are moderately active as germination stimulant
towards seeds of S. hermonthica but remarkably active in
SL mimics the case seeds of Orobanche cernua and Pelipanche
ramosa seeds. A remarkable finding was that introduc-
An interesting and unexpected development is that a tion of an extra methyl group at C-4 gave SL mimics
group of compounds lacking the ABC scaffold also can which were inactive as germination stimulant. This
stimulate germination. These compounds are named as structural change in SL mimics may give a clue for their

Fig. 4 Synthetic analogs of SLs with germinating activity

Fig. 5 Isosteric SL analogs Fig. 6 Debranones and aroyloxy SL mimics

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mode of action. So far the inhibition of shoot branching SLs as inhibitors for shoot branching and in their
of these aroyloxy SL mimics has not been tested, but role in controlling plant architecture
experiments to end this are ongoing. The area of SL
mimics clearly still in its infancy. As mentioned in the introduction, SLs are now recognised
as new plant hormones. An important newly discovered
activity deals with the control of plant architecture. SLs
SLs as branching factors for AM fungi will not operate standing alone, but in concert with other
plant hormones. Until 25 years ago there were 5 types of
A new and important discovery is the role of SLs as plant hormones known, namely: auxins, cytokinins, ethene
branching factors for arbuscular mycorrhizal (AM) fungi (ethylene), gibberellins and abscisic acid (ABA). More
(Akiyama et al. 2005; Parniske 2005, 2008). The struc- recently, brassinosteroids and jasmonates have been added
ture–activity relationship of SLs as branching factors was to the list. The role of the various plant hormones in the
extensively studied by Akiyama et al. (2010), see also plant kingdom is under extensive investigation. There is
Besserer et al. (2006). It was found that (?)-orobanchol accumulating evidence that SLs interplay in a crosstalk
had the highest activity followed by 5-deoxystrigol. (?)- with several of these plant hormones. Which endogenously
GR24 is very active, almost as active as (?)-strigol, but SLs are operative in the interplay in planta is unknown in
its mirror image practically is not (10,000 times less most cases. The crosstalk of SLs with other plant hormones
active). GR7 which is lacking the A-ring is 1000 times may either take place in a fully concerted manner or
less active than GR24 whilst ent GR7 is almost inactive. sequentially in a cascade of events, although in many cases
This information suggests that for an SL analog to be the precise modus operandi is not known in detail.
active as a branching factor for AM fungi all rings of the Phenomenologically, the crosstalk interactions are well
ABC scaffold need to be there and also that the stere- documented.
ochemistry must be as in the strigol family. This implies As it is common for other phytohormones, the SL
that there is not much molecular freedom to design biosynthesis and activity is regulated by other hormones.
simpler structure for interaction with AM fungi. How- For instance, cytokinins act as antagonists to SLs in regu-
ever, it was found recently that the B-ring is not strictly lation of axillary bud outgrowth (Dun et al. 2012) and in
necessary. The SL analogs as shown in Fig. 7 where a regulation of mesocotyl elongation in darkness (Hu et al.
phenyl is connected with the c-carbon of the D-ring or 2014). Auxins are not only shown as one of the major
where a benzyl group is attached to the b-position, both regulators of SL biosynthesis (Hayward et al. 2009; Al-
are appreciably active as branching factors (Akayama, Babili and Bouwmeester 2015, and references therein), but
personal communication, 2015). Both compounds are not also they act as antagonists because SLs may enhance
difficult to prepare as it had been reported previously auxin transport (Cheng et al. 2013, and references therein).
(Nefkens et al. 1997). Lopez-Raez et al. (2010) showed that abscisic acid, one of
Stimulation of AM fungi fulfils a symbiotic role with the key regulators of plant response to abiotic stress, has a
parasitic plants. After the first observation, much attention role in SL biosynthesis, but, on the other hand SLs can also
was given to the beneficial mutualistic and symbiotic impact biosynthesis of abscisic acid (Al-Babili and
associations of AM fungi and parasitic plants (Akiyama Bouwmeester 2015). Besides phytohormones, it is well
and Hayashi 2006; Bonfante and Requena 2011). AM established that phosphate affects SL biosynthesis, mean-
fungi facilitate the uptake of phosphates and nitrates, and ing that shortage of phosphate increases SL production
in a sense these fungi serve as soil fertiliser which may be (Koltai 2015, and references therein).
of agricultural value. Knowledge of this symbiotic rela- However, all these facts are still on cellular level, and
tionship could provide a new strategy for the management they do not explain on a molecular basis which exact
and control of beneficial fungal symbionts and of devas- mechanisms play a role. This is a highly complex research
tating parasitic weeds in agriculture and natural area due to the different effects of phytohormones and
ecosystems. varying context of their actions.

Fig. 7 Simplified SL structures

with activity as a branching
factor for AM fungi

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Most studies on the control of plant architecture are established. Essentially, the truncation method was the
carried out with increased branching mutants, predomi- same as that used for the design of germination stimulants
nantly with ramosus (rms) in garden pea (Pisum sativum), (Zwanenburg and Pospisil 2013). Hence, the design model
more axillary growth (max) in Arabidopsis (Arabidopsis shown in Fig. 3 may also be applicable for shoot branching
thaliana), decreased apical dominance (dad) in Petunia inhibitors. This idea opens new avenues for identifying and
hybrida and dwarf (d) and high tillering dwarf (htd) in rice synthesising new and structurally simple SL analogs for the
(Oryza sativa). Treatment with an exogenous SL, practi- control of plant architecture. Such compounds may be
cally in all cases synthetic GR24 was employed, resulted in potential candidates for agricultural applications.
the inhibition of shoot branching (Dun et al. 2013), stim- It is of interest to note that GR5, the AB-ring truncated
ulation of internode growth (de Saint et al. 2013), accel- analog, strongly inhibits shoot branching when applied
eration of leaf senescence (Yamada et al. 2014), enhance hydroponically, whereas application to the axillary bud of
root hair elongation and the growth of primary roots (Ka- Arabidopsis only gave a weak response. There is a pro-
pulnik et al. 2011), inhibition of the outgrowth of axillary found difference between rice and pea when treated with
buds (Minakuchi et al. 2010), inhibition of formation of a branching controlling inhibitor. In peas a structure–ac-
adventitious and lateral roots (Rasmussen et al. 2012a, b, tivity study for shoot branching, employing the SL-defi-
2013a, b), increasing stem thickness and inducing sec- cient mutant rms1, demonstrated that naturally occurring
ondary growth (Agusti et al. 2011) and other morphologi- SLs, such as 5-deoxystrigol, strigol and orobanchol are all
cal changes. It was found that auxin–SL interactions at highly active but the stereochemistry at C-20 is irrelevant,
multiple levels are critical for branching control (Stirnberg unlike in rice (Boyer et al. 2012, 2014). This was found
et al. 2010; Koltai et al. 2010). How these inhibitory pro- for direct treatment of the axillary buds and in hydroponic
cesses work on a molecular level is still unknown. The culture system. Strigol and orobanchol have a lower
plant physiology and biology of the control plant archi- response than the corresponding acetates probably due to
tecture induced by SLs are beyond the scope of this review. the difference in lipophilicity. A remarkable observation
The relevant details of these aspect of the control of plant was that an extra methyl group at C-30 in GR24 has a
architecture on the cellular level are summarised in several boosting effect on the activity. Unexpectedly, an SL
excellent reviews (Tsuchiya and McCourt 2009; Koltai mimic having an S-aryl at C-20 and an extra methyl group
2011, 2014, 2015; Cheng et al. 2013; Waldie et al. 2014). at C-30 is surprisingly active (Fig. 9). It has not been
The structural requirements are highly relevant for shoot made sure whether the aromatic group in this mimic is a
branching inhibition. The inhibitory effect of a series of 30 prerequisite.
compounds, including the naturally occurring SLs SL mimics (Fig. 9) with an inhibitory effect on shoot
5-deoxystrigol and orobanchol, and the synthetic SL ana- branching of rice mutants d10-1 were reported by the
logs GR24, GR7 and GR5, was investigated with SL-de- Asami group (Fukui et al. 2011, 2013). All these mimics,
ficient rice mutant d10. Some of these compounds were which are also named as debranones (furanones showing
also studied for the effect on Arabidopsis mutant max4 de-branching activity), have O-aryl substituents at C-20 of
(Umehara et al. 2015). This structure–activity study the butenolide ring. Mimics with a Br or a CN group in the
revealed that the R-configuration at C-20 of the D-ring in para position are the most active ones. These compounds
SLs is critical for hormonal activity in rice tillering. This resemble the SL mimic reported by Boyer et al. having an
stereochemistry is present in practically all natural SLs. By S-atom at C-20 . Again, it is not made sure whether the
truncation of the A- and B-ring of the natural SLs the O-aryl group is required for activity. Note that these
minimum structure for activity, involving the D-ring, the debranones are also moderately active as germinating
enol ether moiety conjugated with ester unit (Fig. 8) was agents (see section SL mimics).

Fig. 8 Minimum structure required for shoot branching inhibition in

rice Fig. 9 SLs with an extra methyl group at C-30 in the D-ring

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SLs and karrikinolides (smoke compounds) is necessary. In the early days of SL research, when no
protein structures were available it was tentatively sug-
An intriguing type of compounds was isolated from smoke gested that a nucleophile at the receptor site, for instance an
of bush fires in Australia, which were named karrikins or amino, thiol or hydroxyl group, would react with an SL by
karrikinolides (KARs), after the aboriginal word for an addition–elimination reaction, resulting in a detachment
smoke: ‘karik’ (Flematti et al. 2004; Waters et al. 2012). of the D-ring (Fig. 11) (Mangnus and Zwanenburg 1992).
KARs contains a butenolide ring but its structure features Evidence for this suggested pathway was the isolation of
differ profoundly from that of the butenolide in SLs ABC scaffold product derived from a reaction with ben-
(Fig. 10). zylthiol and benzyl amine.
The KAR structure is planar and achiral, contains two This mechanism was criticised by Scaffidi et al. (2012)
annelated rings, whilst SLs have at least one chiral center, in an attempt to unify the molecular mechanisms of KARs
one of them at the five-membered D-ring. The five-mem- and SLs. Instead of an addition–elimination sequence, an
bered ring in SLs can rotate freely while in KARs it is acyl attack of the D-ring by a nucleophile was proposed as
constraint in a rigid bicyclic system. The KARs contains an an alternative (Fig. 12). For KAR, a reclosure of the five-
exo-methylene group at the c-carbon of the lactone, while membered ring to KAR was proposed (Fig. 12). It should
in SLs this is an acetal type group. It is evident, from the be noted, however, that an acyl attack of an ester is not in
molecular point of view, that the compounds are quite accordance with the generally accepted behaviour of esters,
different entities, each with its own reactivity pattern with thus making this alternative mode of action for SLs less
practically no common features. In spite of this, KARs are likely. Moreover, the isolation of the ABC-adduct (* in
germination stimulants for seeds of Solanum orbiculatum, Fig. 11) obtained by the exposure of GR24 to either ben-
but not for seeds of parasitic weeds (Flematti et al. 2010). zylthiol or benzylamine cannot be explained.
It is perfectly alright to discuss KARs in the same More recently, several studies of protein structures were
context as SLs because they both are germination stimu- reported which shed new light on the signal perception of
lants, albeit for different seed types. However, the justifi- SLs especially in SLs in shoot and branching inhibition
cation that is frequently encountered in the literature, (Hamiaux et al. 2012; Guo et al. 2013; Kagiyama et al.
namely that both stimulants contain a similar butenolide 2013; Nakamura et al. 2013; Zhao et al. 2013). The DAD2
unit is simply not correct. gene was identified from petunia which encodes for an a/b
hydrolase protein DAD2 (Hamiaux et al. 2012). Similarly,
rice genome D14 encodes for the protein D14 (DWARF14)
Mode of action of SLs and a closely related homolog D14-LIKE (D14L)
(Kagiyama et al. 2013; Zhao et al. 2013). The latter is also
The first step in the bioprocesses mediated by SLs involves referred to as KARRIKIN INSENSITIVE 2 (KAI2) present
the interaction of the SLs with a protein receptor. To shed in Arabidopsis and which is specific to karrikins (KARs)
light on this interaction knowledge of the protein structure (Arite et al. 2009). The role of the D14 gene products,

Fig. 10 Comparison of
structural features of SLs and
KARs

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product (probably an artifact) (Hamiaux et al. 2012). On

the basis of this hydrolytic detachment of the D-ring it was
proposed that SLs are essential for the signal transduction,
in spite of the fact that the conditions for this hydrolysis
experiment were far from biomimetic; similarly, incuba-
tion of D14 with GR24 resulting in hydrolysis products
ABC=CHOH and hydroxy butenolide (D–OH) (Zhao et al.
2013; Nakamura et al. 2013). The latter most detailed study
(Nakamura et al. 2013) revealed that the hydrolysis
induced by D14 is stereospecific. (?)-GR24 underwent
hydrolysis much faster than its antipode (-)-GR24. Dif-
ferential scanning fluorimetry (DCF) measurements of
DAD2 with increasing amounts of GR24 indicated a
binding of GR24 with DAD2 in the ratio of 2:1. DCF
Fig. 11 Tentative molecular mechanism for the mode of action via
an addition–elimination reaction measurements were also used to establish the interaction of
SLs (GR24) with the protein D14 (Kagiyama et al. 2013).
Co-crystallisation of GR24 with D14 could not be
accomplished. Zhao et al. (2013) observed in an attempted
co-crystallisation experiment of rice D14 and GR24 an
electron density that was assigned to 2,4,4-trihydroxy-3-
methyl-3-butenal [(HO)2C=C(Me)-CH(OH)-CH=O] which
was proposed as an intermediate en route to hydroxy
butenolide (HO-D). Its formation was rationalised by an
acyl transfer reaction (see Fig. 12, compared Scaffidi et al.
2012, mechanistically not generally accepted behaviour of
esters) involving the D-ring and the serine unit of the
catalytic triad to give the ring-opened product [SerCH2-
OC(=O)C(Me)=CHCH=O] which is then suggested to
undergo a rotation around the olefinic bond (an energeti-
cally highly demanding conversion, unlikely to occur in the
crystal lattice at ambient temperature) to give isomeric
HO2CC(Me)=CH–CH=O. Subsequent addition of water to
the –CH=C(Me)CO2H moiety gives the intermediate
Fig. 12 Tentative molecular mechanism for the mode of action bound to Ser. Lactonization and elimination of water then
involving a nucleophilic attack of ester carbonyl of the D-ring of an results in HO-D. This sequence of events with two ques-
SL (acyl attack, top line). Idem for KAR (bottom line)
tionable steps lacks underpinning and is not an adequate
explanation for the detachment of HO-D without further
whose sequence suggests that they belong to the a/b-fold confirmation.
hydrolase super family, received much attention because A conceivable and more realistic mechanism for the
members of the a/b fold hydrolase superfamily are known detachment of the D-ring is shown in Fig. 13. Bidentate
to participate in hormone signalling for instance those coordination of water fixes the rotation of the D-ring,
involving gibberellin (GA) and the receptor GID1 (Uegu- which leads to a gain of entropy for the reactions to follow.
chi-Tanaka et al. 2005; Murase et al. 2008). The three Water is now favourably disposed for a vinologous water
protein structures, DAD2, D14 and KAI2 are almost addition to the C-ring induced by the Ser unit of the cat-
superimposable, implying that they are orthologs. alytic triad. Subsequent elimination gives HO-D and the
The crystal structure of the protein DAD2 reveals an a/b concurrent formation of the ABC=CHOH fragment. This
hydrolase fold containing a canonical catalytic triad Ser- detachment mechanism is in agreement with the one shown
His-Asp with a large cone-shaped internal cavity capable in Fig. 11 (Nu = H2O).
of accommodating SLs (Hamiaux et al. 2012). The protein Also the SL signalling involves interaction with
was incubated with racemic GR24 in a 1:20 ratio. After PhMAX2A to initiate an SCF-mediated signal transduction
18 h, no GR24 was left and formyl tricyclolactone pathway, but the details were not unveiled by Hamiaux
(ABC=CHOH) resulting from the hydrolysis of GR24 was et al. (2012). The involvement of an a/b-hydrolase in the
isolated by chromatography along with an unknown second SL signal transduction is reminiscent of the GA reception

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Fig. 13 Proposal for the

hydrolysis mechanism of SLs
induced by the catalytic triad of
Ser-His-Asp

system in which the GID1 receptor changes conformation demonstrates that SLs and KARs are entirely different
upon hormone binding (Ueguchi-Tanaka and Matsuoka molecular entities, as already outlined in Fig. 10.
2010). Therefore, the question arises whether D14 and the A receptor protein for the germination of seeds of par-
like exert their action in an analogous manner. The X-ray asitic weeds is not yet available and accordingly no mode
of D14 reveals that the helical lid which is typical for of action on a molecular level can be given. Nevertheless,
GID1, is not present in D14 (Kagiyama et al. 2013). Most we speculate that the protein(s) involved may be (very)
revealing in this respect is the report by the Asami group similar to D14. Support for this hypothesis is that the
(Nakamura et al. 2013; see also Jiang et al. 2013; Zhou bioactiphore for germination, see Fig. 3, has the D-ring
et al. 2013; Koltai 2014; Seto and Yamaguchi 2014). It connected with the C-ring via an enol ether unit which
was convincingly shown that after the detachment of HO- allows the same SL-mediated mechanism with a crucial
D from the SLs by the catalytic action of the Asp-His-Ser role for a canonical catalytic triad and a hydrolytic
triad that this hydroxy butenolide was accommodated in detachment of the D-ring, as shown in Fig. 13. It should be
the active pocket. This induces a conformational change noted that in the model compound for germinating agents,
of the pocket to allow an interaction of D14 and the see Fig. 3, there is a considerable freedom in choosing the
DELLA protein SLR1 resulting in a proteasomal degra- substituent for the A-ring. Striking examples are the ger-
dation in a manner similar to GID1 in GA signalling minating agents with functional labels, see for typical
(Hedden 2008; Murase et al. 2008) allowing the gene examples Fig. 15 (Reizelman et al. 2003; Bhattacharya
transcription to occur. The sequence of events is et al. 2009; Prandi et al. 2011; Rasmussen et al. 2013a, b).
schematically shown in a cartoon (Fig. 14). Remarkably, All these compounds are remarkably active as germi-
introduction of HO-D as such did not induce the D14- nating agents. After hydrolytic detachment of HO-D by
SRL1 interaction. Probably, the ABC moiety serves as a interaction with the protein, ABC=CHOH fragment car-
lipophilic carrier for HO-D. It should be noted that the rying the large substituent will undoubtedly be expelled
ABC=CHOH moiety after being detached from the D-ring from the active cavity. As a consequence, a fluorescent
will be expelled. The stereoselectivity for GR24 can signal was measured upon interaction with a seed of a
readily be explained by the difference in diastereochem- parasitic weed, may be due to the expelled fragment and
ical interactions of (?)-GR24 and (-)-GR24 with the not to the fluorescent SL in the receptor protein. In addi-
chiral walls of the V-shape cavity. tion, protein fishing experiment may be frustrated by the
The interaction of the karrikins (KARs) with KAI2 enzymatic detachment of HO-D and the concurrent
protein was also clarified using an X-ray structure. Inter- removal of the labelled ABC fragment from the protein.
estingly, it was found that the karrikin molecule is not The ‘hook’ in the protein is detached from the ‘fishing line’
hydrolyzed by the protein (Janssen and Snowden 2012; which is expelled.
Guo et al. 2013). The KAR molecule is situated in the The first attempts on the identification of a receptor
opening to the active site close to a helical domain but protein of S. hermonthioca were reported recently (Toh
distal from the canonical catalytic triad of the a/b/hydro- et al. 2015). Using expression in Arabidopsis, it was shown
lase. Without undergoing any molecular change KAR is that ShHTLs [Striga HYPOSENSITIVE TO LIGHT/KAR-
inducing a conformational change in the KAI2 protein RIKIN INSENSITIVE 2 (HTL/KAI2); diverged family of a/
which initiates the signal transduction production process b hydrolase-fold proteins related to D14] might be good
in close analogy to the mode of action of gibberellins. It candidates. However, isolation of a receptor protein from
should be noted that this mode of action of KAR seeds of parasitic weeds has not yet been achieved.

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Planta (2016) 243:1311–1326 1321

Fig. 14 Schematic presentation

of the interaction of an SL with
the receptor protein D14

It remains to fit the activities of the SL mimics shown in in Fig. 6 act as germination stimulants for parasitic weeds
Figs. 6 and 9 into the above patterns for the interactions (Zwanenburg and Mwakaboko 2011; Zwanenburg et al.
with receptor proteins. Seemingly, the mode of action of 2013). Assuming that the mode of action for bud outgrowth
the 5-aroyloxy-substituted butenolides (Fig. 6) can be inhibition and germination of seeds of parasitic weed on
readily rationalised by assuming a hydrolytic removal of the protein level take an analogous pathways for these
the ester unit to give HO-D which then will induce a aroyloxy SL mimics, then this suggested hydrolysis of the
conformational change of the protein pocket in a manner ester substituent cannot account for the blocking effect of a
described above for SLs. Nakamura et al. (2013) indeed methyl group at C-4 of the butenolide ring on the germi-
suggested such a hydrolysis for the interaction of 3,5- nation activity (Zwanenburg et al. 2013). Hence, tenta-
dimethoxybenzoyloxy and anthracene-9-carbonyloxy tively a detour mechanistic pathway is proposed involving
butenolide with D14. These SL mimics inhibit the tiller an initial Michael addition of water to the butenolide,
bud outgrowth in rice. The benzoyloxy butenolides shown followed by an intramolecular transesterification to give

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1322 Planta (2016) 243:1311–1326

Fig. 15 SLs with functional

labels; the bioactiphore is in the
boxed part

the HO-D and the corresponding benzoic acid (Fig. 16). It

is supposed that the Michael addition of water, which is
essential for this pathway, is not possible when a methyl
group is present at C-4. An earlier proposed mechanistic
explanation (Zwanenburg and Pospisil 2013; Zwanenburg
et al. 2013) is not correct as it does not lead to HO-D.
For the debranone type SLs (Fig. 6), a different mode of
action must be operative. This pathway has to account for
the observation that 3,4-dimethyl-5-p-chlorophenylthio-
butenolide (see Fig. 9) is highly active in shoot branching
control (Boyer et al. 2012, 2014). This implies that a
Michael addition of water at C-4 of the D-ring cannot be Fig. 16 Tentative mode of action for SL mimics having an aroyloxy
part of the mode of action on the protein level. substituent at C-5 the D-ring

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Planta (2016) 243:1311–1326 1323

Fig. 17 Fluorescein tagged SL

mimics having a fluorescence
turn-on probe

The aryloxy and arylthia substituent at C-5 of these SL suicidal germination approach. A germination stimulant,
mimics are connected with the D-ring via an acetal type preferably a readily accessible synthetic analog, is applied
unit. Accordingly, a hydrolytic detachment of the HO-D as to the field in the absence of a host. Seed of the weed will
shown for SLs cannot be envisaged. Very recently, highly germinate but due to the lack of nutrients they die. After
relevant new information about SL mimics was reported by that the host plant, usually an important crop, can be
the Tsuchiya et al. (2015). They designed an SL mimic with planted which then does not suffer anymore from the
a fluorescent turn-on probe based on fluorescein (Fig. 17). parasitizing weed (Zwanenburg et al. 2009). Details will be
Conceptually, the design of this sophisticated SL mimic described in a forthcoming review (Zwanenburg, accepted
resembles the ‘‘prodrug approach’’ (Han and Burgess for publication in Pest Manag Sci).
2010): the D-ring tagged with fluorescein reaches the active The recent finding that SLs play an essential role in the
site of the receptor protein whereupon the HO-D is released control of plant architecture led to extensive studies to
to start the signal transduction process. This SL mimic improve the structure of agriculturally important plants.
called Yoshimulactone green (YLG) stimulates germination Details are, however, beyond the scope of this review.
of S. hermonthica seeds with simultaneous release of fluo-
rophoric fluorescein. Practically, all germinated seeds
emitted fluorescence. YLG is almost as active as the stan- Conclusions and future outlook
dard germination stimulant GR24. The hydrolysis products
of YLG were isolated and analyzed by LS/MS. It was The area of strigolactones is rapidly evolving. In recent
shown that YLG binds and acts via ShHTLs, the diverged years much new insight was obtained in the structure and
family of a/b hydrolase-fold protein in S. hermonthica. This bio-properties of naturally occurring SLs, but there is still
protein clearly must be capable of hydrolyzing an acetal much to gain. Reliable models have been developed for the
unit. YLG also interacts with AtD14, the D14 homolog design and synthesis of SL analogs with excellent bio-ac-
derived from Arabidopsis, again releasing fluorescein. This tivity, but further fine tuning is necessary. The SL mimics
elaborate study of Tsuchiya et al. (2015) reveals that SL constitute an important new group of simple compounds
mimics are hydrolyzed by AtD14 and by the ShHTL with a high bio-activity. Further development of SL mimics
receptor proteins in Striga. This hydrolysis is undoubtedly is highly relevant, also in connection with possible appli-
facilitated by the very good leaving ability of the fluorescent cations. Insight into the mode of action has been consid-
probe (Tsuchiya et al. 2015). The debranones shown in erably improved. A consistent picture for SLs, SL analogs
Fig. 6 will follow the same pattern as described for YGL, and SL mimics has been developed, but more information
but the leaving ability of the aryloxy group is less pro- is needed to fully understand the interaction of SLs, its
nounced implying that these debranones are less efficient analogs and mimics with proteins. The role of SLs in
stimulants (Tsuchiya et al. 2015). planta for the control of plant architecture received much
Now it may be concluded that the mode of action of attention and will do so in the years to come. So far the
SLs, SL analogs and SL mimics show a consistent picture protein receptors of seeds of parasitic weeds and AM fungi
in all cases, the release of HO-D is the essential prime have not been isolated and identified; here lies an inter-
trigger for the cascade of reactions leading to the signal esting challenge for the future. The molecular under-
transduction. standing of processes in which SLs play a dominant role is
of utmost importance and may provide new leads for future
research in this exciting area of plant hormones.
Applications of SLs
Author contribution statement BZ (senior author) 60 %,
and both coauthors equal for the remaining part.
SLs, their analogs and mimics have a great potential for
applications in agriculture. The control of parasitic weeds Acknowledgments Part of the research described in this paper is
is under active investigation. One option for this is the imbedded in COST action FA1206 (STREAM). This work was

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supported by the Grant No. L001204 (Sustainable development of Brooks DW, Bevinakatti HS, Powell DR (1985) The absolute
research in the Centre of the Region Haná) from the National Program structure of (?)-strigol. J Org Chem 50:3779–3781. doi:10.
of Sustainability I, MEYS. One of us (BZ) owes many thanks to 1021/jo00220a020
Professor Mirek Strnad for the great hospitality during his stay in the Butler LG (1995) Chemical communication between the parasitic
Department of Growth Regulators, Palacky University, Olomouc weed Striga and its crop host: a new dimension in allelochem-
(CZ). istry. In: Inderji K, Einhellig FA (eds), Insights into allelopathy.
ACS Symposium Series, ACS Books, Washington, DC,
Open Access This article is distributed under the terms of the pp 158–168
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tivecommons.org/licenses/by/4.0/), which permits unrestricted use, occurrence, structure, and biological activity in the rhizosphere.
distribution, and reproduction in any medium, provided you give Phytochem Rev 14:691–711. doi:10.1007/s11101-014-9370-4
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