Exploring the molecular mechanism of karrikins and strigolactones

Article abstract of DOI:10.1016/j.bmcl.2012.04.016

Karrikins and strigolactones are novel plant growth regulators that contain similar molecular features, but very little is known about how they elicit responses in plants. A tentative molecular mechanism has previously been proposed involving a Michael-type addition for both compounds. Through structure-activity studies with karrikins, we now propose an alternative mechanism for karrikin and strigolactone mode of action that involves hydrolysis of the butenolide ring.

Full text of DOI:10.1016/j.bmcl.2012.04.016

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3743–3746
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploring the molecular mechanism of karrikins and strigolactones q
Adrian Scaffidi a, , Mark T. Waters b, Charles S. Bond a, Kingsley W. Dixon c,d, Steven M. Smith a,
⇑
Emilio L. Ghisalberti a, Gavin R. Flematti a
a School of Chemistry and Biochemistry, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
b ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
c School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
d King Park Botanic Garden, Fraser Avenue, West Perth, WA 6005, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Karrikins and strigolactones are novel plant growth regulators that contain similar molecular features,
but very little is known about how they elicit responses in plants. A tentative molecular mechanism
has previously been proposed involving a Michael-type addition for both compounds. Through struc-
ture–activity studies with karrikins, we now propose an alternative mechanism for karrikin and strigo-
lactone mode of action that involves hydrolysis of the butenolide ring.
Received 16 February 2012
Revised 26 March 2012
Accepted 3 April 2012
Available online 10 April 2012
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords:
Karrikinolide
Strigolactones
Plant hormones
Smoke
Molecular mechanism
O
Karrikinolide (KAR1) 1 is a potent seed germination stimulant
derived from burning plant material, and defines a family of
related small molecules known as karrikins (Fig. 1).1,2 These com-
pounds are able to promote seed germination in numerous plant
species native to both fire and non-fire prone regions around the
world.3 Recently, significant attention has been directed towards
this intriguing class of small molecules due to their molecular sim-
ilarity to the strigolactone group of germination and shoot-branch-
ing phytohormones (Fig. 1),4,5 and the fact that both classes of
compounds act through a common cell signalling pathway.5,6
Although the molecular architecture of strigolactones is much
more complex compared to the simpler planar karrikins, both com-
pounds contain substituted methyl butenolide rings and it has
been shown that this functionality is essential for strigolactone
receptor recognition.7,8 Both also contain enol ethers, yet despite
these structural and functional similarities, very little is known
about how these molecules interact with various biomolecules to
elicit a response in plants.
O
C
O
O
A
B
O
O
D
O
O
2
1
Figure 1. Chemical structures of karrikinolide (KAR1) 1 and the synthetic strigo-
lactone (GR24) 2.
nucleophilic addition with thiophenol in a Michael fashion to re-
lease the D-ring and yield a covalent attachment of the nucleophile
to the ABC portion of the molecule.7 Furthermore, the saturation of
the enol ether double bond renders the molecule inactive provid-
ing further evidence that a Michael type addition is required for
bioactivity. Although various labelled derivatives have been pre-
pared to explore this mechanism, no endogenous nucleophile or
receptor molecule has been isolated to date.10 Most recently, Zwa-
nenburg et al. and Fukui et al. report that the enol ether moiety and
ABC rings in strigolactones are not essential for activity, a conclu-
sion based on active analogues lacking both these characteris-
tics.11,12 These analogues replace the enol ether and ABC rings
with a leaving group suggesting that a general labile bond at C5
of the D-ring is sufficient to furnish activity. Furthermore, Zwanen-
burg et al. provide an alternative Michael acceptor mechanism
A proposed mechanism for strigolactone activity has previously
been reported based on the chemistry of the synthetic analogue
GR24 2 (Fig. 2a).7,9 It was demonstrated that GR24 2 undergoes a
q
The authors would like to thank the Australian Research Council (LP0882775,
DP109671) for financial support, Lucy Commander for collecting Solanum orbicul-
atum seed and the Centre for Microscopy, Characterisation and Analysis at UWA.
⇑
Corresponding author.
E-mail address: adrian.scaffidi@uwa.edu.au (A. Scaffidi).
0960-894X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.016

3744
A. Scaffidi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3743–3746
_
O
O
O
O
O
O
a
O
O
O
O
_
Nu
Nu
O
O
O
O
O
O
O
O
O
4
5
6
Figure 4. Chemical structures of saturated karrikin analogues.
O-
O
O-
O
O
O
O
O
+
Nu
analogues have recently been reported and were prepared accord-
ing to published procedures, with the exception of the tetrahydro-
pyran derivative 4.14 We found that catalytic hydrogenation of 1
resulted in clean conversion to 4 in good yield.
_
O
O
b
The biological evaluation of 5 as a germination stimulant has
been reported,15 but not in direct comparison with KAR1 1. There-
fore, the ability of 4, 5,16 and 617 to promote seed germination was
evaluated using Solanum orbiculatum seeds that had low germina-
tion in water controls (<10%) and were stimulated to over 90% ger-
O
H+
O
_
O
Nu
O
Nu
mination with 1.18 Our results show that in the range of 1–10
lM,
O
both 516 and 617 were able to stimulate germination up to 100%,
however, similar germination response was achieved with much
lower concentrations of 1 (Fig. 5). In contrast, the fully saturated
pyran analogue 4 only promoted germination at the highest con-
centration tested, and achieved only 60% germination. Hence, the
removal of the conjugated enol ether functionality in KAR1 still
renders the molecule active albeit with reduced potency.7
O
O
O
O
Nu
HO
Nu
Figure 2. Previously proposed mechanisms for the Michael addition of a nucleo-
phile to karrikins and strigolactones.
To explore the functional importance of the 4H-pyran ring, we
prepared a number of conjugated butenolide rings with either a
terminal alkene or enol ether functionality (Fig. 6). All the monocy-
clic analogues 7–10 failed to stimulate germination at all concen-
based solely on the D-ring.12 In light of these results and given that
the Michael acceptor mechanism has also been considered feasible
for karrikins (Fig. 2b),4,9 we investigated the ability of KAR1 1 to un-
dergo nucleophilic addition, and explored the bioactivity of various
saturated analogues.
trations tested (1 nM–10 lM, data not shown). This result
implies that a 4H-pyran ring is essential for maximum karrikin
activity and that the mode of action does not proceed through a
Michael type addition at either C5 or C7. The exact function of
the 4H-pyran ring remains unclear, but we postulate that the un-
ique electronics associated with the extended conjugation present
in the KAR1 backbone are responsible for determining the extent to
which the molecule stimulates germination. Evidence to support
this hypothesis comes from the limited activity of the difluoro-
methyl derivative 1119 as previously observed20 (Fig. 6). The elec-
tron-withdrawing capacity of the difluoromethyl group, which is
similar in size to a methyl group, renders the compound inactive
in promoting seed germination.
To provide evidence for a nucleophilic addition mechanism
in vivo, initially we sought to determine whether KAR1 1 is a suit-
able Michael acceptor. KAR1 1 contains two potential sites on the
4H-pyran ring (C5 and C7) that could undergo nucleophilic addi-
tion in a Michael type mechanism making outcome prediction dif-
ficult.4,9 Our first approach followed the methods of Kupchan et al.
who demonstrated the addition of cysteine to a number of alkyl
substituted butenolides.13 Although cysteine represents a biologi-
cally relevant nucleophile, no addition products were detected
using this method. We then investigated whether the use of a
stronger nucleophile, such as benzyl mercaptan, would result in
an addition product. As expected, KAR1 was consumed with the
proposed enol trapped by treatment with acetic anhydride to fur-
nish the acetate 3. This confirmed the ability of KAR1 1 to undergo
a Michael type addition exclusively at C5 (Fig. 3). To investigate
whether this mechanism is required to stimulate germination, a
number of reduced KAR1 derivatives were prepared and their bio-
activity evaluated.
The above observations indicate an alternative mechanism is
required to rationalise the activity of karrikins. Recently, we iden-
tified from Arabidopsis thaliana two genes, KARRIKIN INSENSITIVE 2
The three primary targets were analogues containing various
saturation patterns on the 4H-pyran ring (Fig. 4). All three
O
O
O
i. BnSH, NaH, THF
ii. Ac2O, pyridine
O
O
AcO
SBn
1
3
Figure 5. Germination of Solanum orbiculatum seeds tested with various saturated
Figure 3. Nucleophilic addition to KAR1 1.
karrikin analogues.

A. Scaffidi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3743–3746
3745
O
O
O
O
O
O
a
CHF2
O
O
_
O
O
O
O
O
O
O
Nu
_
+
O
Nu
O
O
MeO
O
EtO
O
O
7
8
9
10
11
2
12
Figure 6. Chemical structures of butenolide analogues that possess a Michael
acceptor similar to karrikinolide but are not active as seed germination stimulants.
HO
O
O
OH
+ H2O
- NuH
O
O
b
O
O
O
_
Nu
H+
Nu
Nu
O
HO
O
O
O
O
13
O
O
O
HO
O
- H2O
+ H2O
- NuH
O
O
Figure 7. KAR1 (green) is predicted to fit well into the large active site hydrophobic
pocket of KAI2 (grey sticks and surface), in a similar manner to PMSF (blue) in RsbQ.
The homology model of the active site of KAI2 (SWISS-MODEL)24 is based on the
structure of RsbQ complexed with PMSF (PDB entry 1WPR).22 The active sites of
KAI2 and RsbQ are almost identical, with only one of the amino acids pictured here
being different (Ala to >Ile). Figure prepared with PyMOL.25 KAR1 has been
manually posed over PMSF to illustrate similar relative positions of the electrophilic
carbon and six-membered ring.
Figure 8. New proposed molecular mechanism for (a) strigolactones and (b)
karrikins involving the hydrolysis of the butenolide ring.
metabolite. Such a mechanism is consistent with observations
from RsbQ, whereby upon binding of a small molecule in the active
site, the cap domain of the protein is stabilised possibly permitting
the RsbP phosphatase to bind.22 If this scenario also applies to
KAI2, the product of hydrolysis of the karrikin butenolide stabilised
in the active site could trigger a new protein conformation that
would only revert when the product is subsequently ejected from
the active site. In this regard, it may be useful to note that the
hydrolysis of karrikins generates the intermediate 13 which has
similarity to the intermediate 12 that is generated upon strigolac-
tone hydrolysis (Fig. 8).
In conclusion, significant attention has been directed towards
strigolactone phytohormones given their ability to promote seed
germination,27 regulate shoot branching in plants28,29 and stimu-
late arbuscular mycorrhizal fungi associations with plant roots.30
Similarly, the ability of karrikins to promote seed germination is
also attracting worldwide attention,4,31 and it is clear that strigo-
lactones and karrikins have structural and functional similari-
ties.5,32 Despite the importance of these bioactive molecules in
multiple facets of plant development, very little is known about
their mode of action. Based on karrikin structure–activity relation-
ships, we propose a new mechanism for the activity of karrikins
and strigolactones involving hydrolysis of the butenolide ring. This
work will help direct future endeavours to elucidate the mode of
action for both classes of bioactive compounds and will lead to
the development of simpler more potent germination stimulants
and plant growth regulators.
(KAI2) and AtDWARF14 (AtD14), that mediate the distinct percep-
tion of karrikins and strigolactones, respectively.6 Both encode pro-
teins belonging to a diverse family of
a/b hydrolases which are
distinguished by a highly conserved catalytic triad composed of
histidine, serine and aspartic acid residues.6,21 The only member
of this family to be well characterised is RsbQ from the bacterium
Bacillus subtilis.22,23 The crystal structure of RsbQ reveals the cata-
lytic triad residing within a cavity that is predicted to bind a small
hydrophobic molecule as a substrate for hydrolytic activity.22 Fur-
thermore, the serine protease inhibitor phenylmethanesulfonyl
fluoride (PMSF) has been shown to label the catalytic triad, and it
is possible to overlay KAR1 1 on PMSF to achieve a similar align-
ment in silico (Fig. 7).
Accordingly, we tentatively propose that KAI2 and AtD14 might
hydrolyse the butenolide rings of karrikins and strigolactones,
respectively. In the case of strigolactones, this would result in the
cleavage of the ABC-rings from the D-ring analogous to the prod-
ucts resulting from a Michael addition (Fig. 8a). This single mech-
anism can account for all active strigolactone analogues including
derivatives lacking the enol ether functionality, but possessing a
leaving group attached to C5 of the D-ring. With regards to karri-
kins, the butenolide hydrolysis is difficult to rationalise as the
hydrolysis product would furnish a ketone that would favour karr-
ikin reformation upon dehydration with the resulting acid (Fig. 8b).
However, given that both the ABC and D-rings of strigolactones
themselves are not active,26 we propose that the act of hydrolysis
of the butenolide ring of both germination stimulants may provide
an intracellular signal, potentially as a result of a change in protein
tertiary structure rather than due to the formation of an active
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
016.

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A. Scaffidi et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3743–3746
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