Tryptophan derivatives regulate the seed germination and radicle growth of a root parasitic plant, Orobanche minor

Article abstract of DOI:10.1016/j.bmcl.2021.128085

Root parasitic plant germination is induced by the host-derived chemical, strigolactone (SL). We found that a major microbial culture broth component, tryptone, inhibits the SL-inducible germination of a root parasitic plant, Orobanche minor. L-tryptophan (L-Trp) was isolated as the active compound from tryptone. We further found that L-Trp related compounds (1b-11), such as a major plant hormone auxin (8, indole-3-acetic acid; IAA), also inhibit the germination and post-radicle growth of O. minor. We designed a hybrid chemical (13), in which IAA is attached to a part of SL, and found that this synthetic analog induced the germination of O. minor, and also inhibited post-radicle growth. Moreover, contrary to our expectations, we found that N-acetyl Trp (9) showed germination stimulating activity, and introduction of a substitution at C-5 position increased its activity (12a-12f). Our data, in particular, the discovery of a structurally hybrid compound that has two activities that induce spontaneous germination and inhibit subsequent radical growth, would provide new types of germination regulators for root parasitic plants.

Full text of DOI:10.1016/j.bmcl.2021.128085

Bioorg. Med. Chem. Lett. 43 (2021) 128085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Tryptophan derivatives regulate the seed germination and radicle growth of
a root parasitic plant, Orobanche minor
Michio Kuruma, Taiki Suzuki, Yoshiya Seto*
Department of Agricultural Chemistry, School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki-shi, Kanagawa 214-8571, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Root parasitic plant germination is induced by the host-derived chemical, strigolactone (SL). We found that a
major microbial culture broth component, tryptone, inhibits the SL-inducible germination of a root parasitic
plant, Orobanche minor. L-tryptophan (L-Trp) was isolated as the active compound from tryptone. We further
found that L-Trp related compounds (1b-11), such as a major plant hormone auxin (8, indole-3-acetic acid; IAA),
also inhibit the germination and post-radicle growth of O. minor. We designed a hybrid chemical (13), in which
IAA is attached to a part of SL, and found that this synthetic analog induced the germination of O. minor, and also
inhibited post-radicle growth. Moreover, contrary to our expectations, we found that N-acetyl Trp (9) showed
germination stimulating activity, and introduction of a substitution at C-5 position increased its activity (12a-
12f). Our data, in particular, the discovery of a structurally hybrid compound that has two activities that induce
spontaneous germination and inhibit subsequent radical growth, would provide new types of germination reg-
ulators for root parasitic plants.
Root parasitic plant
Radicle growth
Strigolactone
N-acetyl tryptophan
IAA
Introduction
way to remove the parasitic plant seeds from infected fields. However,
for practical applications, large amounts of the SL molecules need to be
Root parasitic plants in the family Orobancheceae, such as Striga and
Orobanche, cause significant damage to major crops, such as sorghum,
millet, maize, sugarcane and upland rice, particularly in sub-Saharan
Africa. The annual loss caused by these parasitic plants is estimated to
be billions of US dollars. The seed germination of these root parasitic
plants is induced by host-derived chemicals collectively called strigo-
lactones (SLs). This germination system is thought to be a survival
strategy in which the parasitic plants germinate only in the host’s
presence, because they cannot survive without attaching to the host. In
2005, SLs were re-characterized as symbiotic signals for arbuscular
mycorrhizal fungi that aid in supplying inorganic nutrients, such as
phosphate, to host plants1. Moreover, in 2008, it was discovered that SLs
prepared and spread on the agricultural fields. Because the amounts of
naturally existing SLs are extremely small, at present, chemical synthetic
approaches have been used, and SL analogs, such as the commonly used
GR24, have been developed. In most cases, these synthetic analogs
contain a methylbutenolide moiety (D-ring), which is a common
component of natural SL molecules. The attachment of the D-ring to
various structures has resulted in yielding many SL analogs4. Recently,
the SL receptors in a parasitic plant, Striga hermonthica, were identi-
fied5–7, which greatly facilitated the screening of chemicals that target
these receptor proteins. Using this approach, a femtomolar-range suicide
germination inducer, sphynolactone-7, which also has the D-ring moi-
ety, was developed recently8. In contrast, there have been limited ex-
amples of structurally SL-unrelated chemicals that have SL-like
bioactivities.
act as a novel class of plant hormones that regulate shoot branching2,3
.
Parasitic plants typically produce a very large number of extremely
tiny (approximately 0.2 mm) seeds that may remain dormant for de-
cades. Once the seeds detect the SL molecules emitted from host roots,
they are released from dormancy, germinate and penetrate into the host
roots. However, if the parasitic plant seeds germinate in the absence of a
host, then they die within 5 days. A method called ‘suicidal germination’
strategy was based on this observation and is thought to be an effective
In addition to these chemical synthetic or chemical biological ap-
proaches, producing SLs or chemicals with the same bioactivities in
microorganisms may be useful. Some plant pathogenic microorganisms
produce plant hormone molecules as secondary metabolites. For
instance, Gibberella fujikuroi, the causal fungus of rice ‘bakanae’ disease,
produces the plant hormone gibberellin (GA), and this fungus is
* Corresponding author.
E-mail address: yoshiya@meiji.ac.jp (Y. Seto).
https://doi.org/10.1016/j.bmcl.2021.128085
Received 23 February 2021; Received in revised form 22 April 2021; Accepted 29 April 2021
Available online 6 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

M. Kuruma et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128085
culture broth on O. minor’s seed germination to determine whether
culture broth alone affects germination. During this process, we
discovered that a major culture broth component, tryptone, which is
often used as a nitrogen source, strongly inhibited the GR24-inducible
germination of O. minor seeds in a dose-dependent manner (Fig. 1A).
We performed a reversed-phase high performance liquid chromatog-
raphy (HPLC) analysis of tryptone and detected the inhibitory activity in
a probable single-peak fraction (Fig. S1). Therefore, this fraction was
collected, and the structure of the isolated chemical was determined to
be a major amino acid, L-tryptophan (L-Trp) (Fig. 1B). In a previous
report, the effects of 20 amino acids on the germination and radicle
growth of O. minor seeds were determined, and several amino acids,
including L-Trp, inhibited both the seed germination and radicle growth
of O. minor9. Thus, our data confirmed these reported results.
To understand the structural requirements for the inhibition of
germination and radicle growth by L-Trp, we tested some commercially
available Trp-related chemicals. Commercial L-Trp inhibited O. minor
germination, but the inhibitory activity of its enantiomer, D-Trp (1b) was
relatively weak. Because some derivatives which have a substitution at
C-5 position were relatively inexpensive to purchase, we tested the ac-
tivity of such analogs. The introduction of a methyl group at the C-5
position (2) produced a weaker activity than L-Trp, whereas the intro-
duction of a hydroxyl group at the same position (3) increased the ac-
tivity compared to L-Trp (Fig. 2). Tryptamine (4) and 5-OH-tryptamine
(5) showed weaker activity levels compared with L-Trp. L-Trp methyl
ester (6) and L-tryptophanol (7) showed almost the same activity level as
L-Trp (Fig. 2). In planta, indole-3-acetic acid (IAA; 8), a plant hormone is
biosynthesized using Trp as a biosynthesis intermediate. Interestingly,
the germination-inhibitory activity of IAA was the strongest among all
the tested chemicals. In addition, L-Trp and some of the tested chemicals
inhibited radicle elongation (Fig. S2)9, and among of them, IAA showed
Fig. 1. Effect of typrotone on the GR24 induced germination of O. minor seed.
A. Germination rate of O. minor seeds after co-treatment of GR24 and different
concentration of tryptone. Cont. indicates distilled water with 0.1% acetone.
Data are the means ±SD (n=3). B. Chemical structure of isolated
active compound.
currently used to fermentatively produce GA. GA may be used as an
agrochemical, which can, in particular, generate seedless grapes.
Thus, microorganisms that can produce SLs or chemicals having SL-
like bioactivities should be used to quantitatively prepare suicidal
germination inducers for root parasitic plants. Consequently, we
decided to screen for microorganisms that have the ability to produce
chemicals with seed germination-inducing activities for the root para-
sitic plant, Orobanche minor. Before establishing an efficient screening
method, we evaluated the effects of components derived from microbial
Fig. 2. Effects of tryptophan-related compounds on O. minor germination. A.Chemical structures of tested compounds. B. Germination percentage of O. minor seed
after co-treatment of GR24 (1
μ
M) and tested compounds at indicated concentration. Data are the means ±SD (n=4).
2

M. Kuruma et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128085
Fig. 3. Germination stimulating activity of N-acetyl-tryptophan derivatives for O. minor. A. Germination stimulating activity of L-Trp related compounds at 1 mM (4,
8, 10, 11) or 2.5 mM (1–3, 5–7, 9). Data are the means ±SD (n=3).B. Chemical structures of tested compounds. C. Germination percentage of O. minor seed after
treatment with tested compounds at indicated concentrations. Data are the means ±SD (n=4).
the strongest inhibitory effect.
growth inhibitory activities. This type of hybrid chemical had been
synthesized previously and had reported germination-inducing activ-
ities for several root parasitic plants, although the inhibitory activity for
radical growth was not evaluated.13Consistent with our expectations,
the synthesized compound 13 induced O. minor germination at moder-
ately low concentrations and radicle growth inhibition was observed for
post-germination seeds (Fig. 4B, C; Fig. S4). Thus, we could claim that
compound 13 was the first case to develop a compound having the both
activities. It is still unclear whether IAA was released in planta by
receptor-dependent hydrolysis of compound 13, nonspecific hydrolysis
or nonenzymatic degradation. However, if this compound can suppress
parasitism by inhibiting post-germination radicle growth, then such
chemical can be used as a suicidal germination inducer even in the
presence of host plants.
N-Acetyl-L-Trp (9), N-formyl-DL-Trp (10) and a dipeptide derivative,
Glycyl-DL-Trp (11), which all have a substitution in the amino group,
showed very weak germination-inhibitory activities against O. minor
(Fig. 2B). In contrast, to our surprise, 2.5 mM N-acetyl-L-Trp very weakly
induced the germination of O. minor (Fig. 3A). The activity was much
weaker than that of GR24, but it was reproducible. Additionally, we
chemically synthesized some derivatives of N-acetyl-Trp having
different substitutions at the C-5 position, because a starting material,
indole, which has a different substitution at C-5 position is available at a
relatively low price (Fig. 3B, 12a–f). Interestingly, all the C-5-substituted
N-acetyl-Trp molecules showed stronger germination-stimulating ac-
tivities than non-substituted N-acetyl-Trp (Fig. 3B, C). Although these
activity levels were still weaker than that of GR24, these chemicals,
which are not structurally related to SL, do represent new types of
germination inducers for root parasitic plants.
We identified L-Trp (1), which is contained in the major microbial
culture broth component tryptone, as a germination inhibitor of
O. minor. Thus, culture broth components in a bioassay-guided screening
using O. minor seeds must be carefully selected because the components
of the medium itself might exhibit inhibitory activity. Some Trp-related
compounds exhibited similar germination-inhibitory activities and also
inhibited radicle elongation. The activity of a plant hormone, IAA, was
strongest among all the tested chemicals. In addition, it was previously
reported that IAA treatments inhibit infection by the parasitic plant
Orobanche aegyptiaca14. IAA is well known to regulate root growth of
seed plants with inhibitory effect on main root growth at high concen-
tration. Therefore, other tested chemicals may also be converted into
IAA in planta, resulting in the inhibition of germination and radicle
growth. On the contrary, some of the N-acetylated Trp derivatives
showed germination-inducing activities for O. minor. The chemical
synthesis of some analogs having substitutions at the C-5 position pro-
vided moderately active compounds; however, their activity levels were
weaker than that of GR24. Most of the reported germination inducers for
root parasitic plants are structural derivatives of SLs. Although the
As stated above, IAA and some Trp-related chemicals inhibited not
only the germination, but also the post-germination radicle growth, of
O. minor. In planta, SLs are perceived by an α/β-fold hydrolase family
protein receptor, such as DWARF14 or HYPOSENSITIVE TO LIGHT/
KARRIKIN INSENSITIVE2 (HTL/KAI2: hereafter referred to as HTL)10
.
In the parasitic plant S. hermonthica, some HTL family proteins have
been identified as SL receptors required for germination5–7. Although
the SL receptors in O. minor have not been identified, Striga HTL ho-
mologs are also conserved in O. minor5. In addition, DWARF14 and Striga
HTL proteins possess hydrolytic activities with the SL ligand mole-
cule7,11,12. In the SL structure, the D-ring is the most critical component
for the bioactivities of SL-related chemicals. Even when a molecule that
is structurally unrelated to SLs is attached to the D-ring, these synthetic
analogs often show SL-like bioactivities4. Consequently, we chemically
synthesized a hybrid SL analog in which the D-ring moiety was attached
to the carboxylic acid part of IAA (Fig. 4A, 13) to determine whether one
compound might have both seed germination-inducing and radicle
3

M. Kuruma et al.
Bioorganic & Medicinal Chemistry Letters 43 (2021) 128085
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was funded by JSPS KAKENHI (Grant No. 19K05852), the
Kato Memorial Bioscience Foundation and the Mitsubishi Foundation.
We thank Dr. Xiaonan Xie for providing the O. minor seeds. We thank
Lesley Benyon, PhD, from Edanz Group (https://en-author-services.
edanz.com/) for editing a draft of this manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128085.
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4