Identification of methoxylchalcones produced in response to CuCl2 treatment and pathogen infection in barley

Article abstract of DOI:10.1016/j.phytochem.2020.112650

Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl₂ solution as an elicitor. LC-MS analysis of the CuCl₂-treated leaves showed the induced accumulation of three compounds. Among them

Full text of DOI:10.1016/j.phytochem.2020.112650

Phytochemistry 184 (2021) 112650
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Identification of methoxylchalcones produced in response to CuCl2
treatment and pathogen infection in barley
Naoki Ubea, Yuhka Katsuyamab, Keisuke Kariyac, Shin-ichi Tebayashid, Masayuki Suee,
,
*
Takuji Tohnookaf, Kotomi Uenob, Shin Taketag, Atsushi Ishiharab
a Arid Land Research Center, Tottori University, Tottori, 680-8553, Japan
b Faculty of Agriculture, Tottori University, Tottori, 680-8553, Japan
c Graduate School of Sustainability Science, Tottori University, Tottori, 680–8553, Japan
d Faculty of Agriculture and Marine Science, Kochi University, Monobe, Nankoku, Kochi, 783–8502, Japan
e Department of Agricultural Chemistry, Tokyo University of Agriculture, Tokyo, 243-0034, Japan
f National Agriculture and Food Research Organization, Tsukuba, 305-8518, Japan
g Institute of Plant Science and Resources, Okayama University, Kurashiki, 710-0046, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl2 solution
as an elicitor. LC-MS analysis of the CuCl2-treated leaves showed the induced accumulation of three compounds.
Among them, two were purified by silica gel and ODS column chromatography and preparative HPLC and were
identified as 2ʹ,3,4,4ʹ,6ʹ-pentamethoxychalcone and 2ʹ-hydroxy-3,4,4ʹ,6ʹ-tetramethoxychalcone by spectroscopic
analyses. The remaining compound was determined as 12-oxo-phytodienoic acid (OPDA), a major oxylipin in
plants, by comparing its spectrum and retention time from LC-MS/MS analysis with those of the authentic
compound. The accumulation of these compounds was reproduced in leaves inoculated with Bipolaris sor-
okiniana, the causal agent of spot blotch of the Poaceae species. This inoculation increased the amounts of other
oxylipins, including jasmonic acid (JA), JA-Ile, 9-oxooctadeca-10,12-dienoic acid (9-KODE), and 13-oxoocta-
deca-9,11-dienoic acid (13-KODE). The treatments of the barley leaves with JA and OPDA induced the accu-
mulation of methoxylchalcones, but treatment with 9-KODE did not. These methoxylchalcones inhibited conidial
germination of B. sorokiniana and Fusarium graminearum, thereby indicating that these compounds possessed
antifungal activity. Consequently, they are considered to be involved in the chemical defense processes as
phytoalexins in barley. Accumulation of methoxylchalcones in response to JA treatment was observed in all
seven barley cultivars tested, but was not detected in other wild Hordeum species, wheat, and rice, thus indi-
cating that their production was specific to cultivated barley.
Hordeum vulgare
Poaceae
Phytoalexin
Methoxylchalcone
Flavonoid
Pathogen infection
1. Introduction
demonstrated
by
various
experiments.
For
example,
a
phytoalexin-deficient mutant (pad3) was shown to be more susceptible
to pathogenic infection than phytoalexin-producing wild type plants in
Arabidopsis thaliana (L.) Heynh. (Thomma et al., 1999). Genetically
modified tobacco (Nicotiana tabacum L.) plants that accumulate a
foreign phytoalexin, resveratrol, showed enhanced resistance to the gray
mold fungus Botrytis cinerea Persoon: Fries (Hain et al., 1993).
Plants employ various defense mechanisms to prevent pathogen
infection, including phytoalexin accumulation. Phytoalexins are low-
molecular-weight, anti-microbial compounds produced by plants in
response to pathogen infection, and their accumulation leads to the
formation of an inducible chemical barrier against pathogen attacks.
The accumulation of phytoalexins is induced by oxylipins such as jas-
monic acid, heavy-metal ions, and UV irradiation, as well as pathogen
infection (Nojiri et al., 1996; Rakwal et al., 1996; Kato et al., 1994;
Schmelz et al., 2011). The significance of phytoalexin accumulation in
the interaction between plants and pathogenic microorganisms has been
Flavonoids are widely distributed specialized metabolites in the
plant kingdom. They are biosynthesized from L-phenylalanine via the
phenylpropanoid pathway in common with other phenolic metabolites.
The first committed step in the biosynthetic pathway is a reaction
catalyzed by chalcone synthase (EC 2.3.1.74), where one molecule of
* Corresponding author.
E-mail address: aishihara@tottori-u.ac.jp (A. Ishihara).
https://doi.org/10.1016/j.phytochem.2020.112650
Received 14 September 2020; Received in revised form 8 December 2020; Accepted 23 December 2020
Available online 30 January 2021
0031-9422/© 2021 Elsevier Ltd. All rights reserved.

N. Ube et al.
Phytochemistry 184 (2021) 112650
hydroxycinnamoyl-CoA is condensed with three molecules of malonyl-
CoA to form a chalcone, which is subsequently converted to flavanone
by chalcone isomerase (EC 5.5.1.6.). Flavonoids can be extensively
modified through hydroxylation, O-methylation, and C- or O-glycosyl-
ation, depending on the plant species, organs, and development stages.
Flavonoids provide UV protection and pigmentation, protect against
pathogens and pests, promote pollen fertility, communicate with mi-
croorganisms, and regulate auxin transport (Winkel-Shirley, 2001).
Some flavonoids contribute to the inducible, chemical defense re-
sponses as phytoalexins. Sakuranetin (7-O-methylnaringenin) is a
flavonoid phytoalexin in rice (Oryza sativa L.) and is crucial for disease
resistance because of its strong antifungal activity against pathogens
(Kodama et al., 1992; Hasegawa et al., 2014; Murata et al., 2020).
3-Deoxyanthocyanidins are flavonoid phytoalexins in sorghum (Sor-
ghum bicolor (L.) Moench) (Nicholson et al., 1987), and their accumu-
lation is stimulated by inoculation with either pathogenic or
non-pathogenic fungi in sorghum (Nicholson et al., 1987; Snyder and
Nicholson, 1990). When the yellow seed1 (y1) allele that encodes the
transcription factor MYB loses its functionality, 3-deoxyanthocyanidine
accumulation decreases and the susceptibility to sorghum pathogens is
enhanced (Ibraheem et al., 2010).
2. Results
2.1. Detection of inducible metabolites in barley leaves treated with CuCl2
Barley leaves were treated with a CuCl2 solution by the floating
method, and the effects of the treatment on specialized metabolite
accumulation were analyzed. The CuCl2 solution was used as an elicitor
because it has been shown to be effective in inducing the accumulation
of specialized metabolites in various plants (Rakwal et al., 1996; Oikawa
et al., 2001; Ube et al., 2019a). The third leaves of 3-week-old barley
seedlings were treated with a 0.5 mM solution of CuCl2 because this
treatment effectively induced the accumulation of phytoalexin tritica-
mides A and B in wheat (Ube et al., 2019a,b), and the metabolites in the
leaves were extracted with 80% methanol 72 h after commencement of
the treatment. The extracts were subjected to liquid chromatography
mass spectrometry (LC-MS) analysis. We manually checked ions corre-
sponding to the peaks detected in the total ion chromatograms (TICs,
Fig. 1), and generated selected ion chromatograms (SICs). In the SICs,
other than the peaks of known phytoalexins, triticamides A (m/z 303
[M-OH]+, retention time: 6.75 min) and B (m/z 305 [M+H]+, retention
time: 7.22 min), only three SIC peaks of the ions at m/z 359, 293, and
345 showed prominent increases in the peak area in the CuCl2-treated
leaves as compared to the peaks in control leaves. The compounds
corresponding to these peaks were referred to as 1–3. Triticamide C was
not detected under these analytical conditions due to its small accu-
mulated amount. Although other peaks showed slightly increased in
peak area in the SICs, their fold changes were less than 3-fold.
Oxylipins such as jasmonic acid (JA) and 12-oxo-phytodienoic acid
(OPDA) have been shown to induce defensive responses in plants.
“Oxylipins” are oxidized fatty acids with unique physiological roles.
Specific oxylipins function as plant signaling molecules that regulate
diverse processes such as development, reproduction, stress acclimation,
and innate immune responses against pests and pathogens (Farmer
´
et al., 2003; Wasternack and Hause, 2013; Almeras et al., 2003).
Recently, the accumulation of 9-oxooctadeca-10,12-dienoic acid
(9-KODE) and 13-oxooctadeca-9,11-dienoic acid (13-KODE) was shown
to be induced in response to JA treatment and pathogen infection in rice
leaves (Nishiguchi et al., 2018). Furthermore, JA and these KODEs
induced defensive metabolites, such as sakuranetin and serotonin, in
rice leaves. In wheat (Triticum aestivum L.), JA treatment led to accu-
mulation of wheat phytoalexins, triticamides A and B (Ube et al.,
2019a). The production of maize (Zea mays L.) phytoalexins, such as
kauralexins and zealexins, is stimulated by co-treatment with JA and
ethylene (Schmelz et al., 2011; Huffaker et al., 2011).
2.2. Identification of 1 and 3
We purified 1 and 3 from barley leaves treated with 0.1 mM CuCl2 for
72 h. The molecular formula of 1 was found to be C20H22O6, as deter-
mined by HR-MS (m/z 359.1487 [M+H]+). The 1H NMR spectrum of 1
(Table 1, Fig. S1) showed two groups of aromatic proton signals: one at
6.17 ppm represented a tetrasubstituted benzene ring, and the other had
peaks at 6.85, 7.06, and 7.09 ppm, corresponding to a trisubstituted
benzene ring. Additionally, the spectrum revealed the coupling of two
double-bond protons (6.84 and 7.28 ppm, J = 16.2 Hz) and the presence
of five methoxy groups (3.77–3.91 ppm). Considering the molecular
formula and partial structures, a carbonyl group was assumed to be
present. The 1H–1H COSY spectrum showed a correlation between the
double-bond proton signals and those of the trisubstituted benzene ring
(Fig. 2A and Fig. S2). These partial structures suggested that 1 possesses
a chalcone skeleton. Five methoxy groups were considered to be bound
to the two benzene rings of this skeleton because one ring had three
positions and the other two positions were available for substitution by
methoxy groups. In the 1H–1H COSY spectrum, three methoxy groups at
3.77 and 3.86 ppm showed correlations to aromatic protons at 6.17 ppm
in the tetrasubstituted ring, while two methoxy groups at 3.91 and 3.90
ppm showed correlations to aromatic protons at 6.85 and 7.06 ppm in
the trisubstituted ring, respectively. Two protons of the tetrasubstituted
benzene ring were observed as one signal at 6.17 ppm, indicating that
this benzene ring possessed a symmetric structure, giving either a 2,4,6-
or 3,4,5-trimethoxybenzene ring. The relatively high magnetic field shift
of this signal suggested that it was likely a 2,4,6-trimethoxybenzene
ring. The three proton signals from the trisubstituted benzene ring
appeared at 6.85, 7.06, and 7.09 ppm and indicated occurrence of ortho
and meta coupling. Considering their chemical shifts and coupling pat-
terns, the trisubstituted benzene ring could possess three protons at
positions 2, 5, and 6 or positions 2, 4, and 5. Based on the 1H NMR
spectra of pentamethoxychalcones from previous reports, we found the
spectrum of 1 to be identical to that of 2ʹ,3,4,4ʹ,6ʹ-pentamethox-
ychalcone (PMC, Chu et al., 2004). Thus, the identity of 1 was deter-
mined and the structure has been shown in Fig. 2B.
Changes in flavonoid metabolism in response to exogenous appli-
cation of JA have been found in barley (Hordeum vulgare L., Poaceae).
The barley plant constitutively accumulates the C-glycosylated flavones
isovitexin-7-O-glucoside (saponarin) and isoorientin-7-O-glucoside
(lutonarin) during primary leaf development (Reuber et al., 1996).
Saponarin was shown to be modified by acylation to generate 6ʹʹʹ-sina-
poyl-saponarin, 6ʹʹʹ-feruloyl-saponarin, and 4ʹ-glucosyl-6ʹʹʹ-sinapoyl-sa-
ponarin in JA-treated young leaves (Ishihara et al., 2002).
Recently, we observed an increased accumulation of the dimer of
serotonin and 3-(2-aminoethyl)-3-hydroxyindolin-2-one after infection
with Bipolaris sorokiniana (Sacc. ex Sorok.) Shoem. and CuCl2 treatment
in barley leaves and proposed that they served as phytoalexins in barley
(Ishihara et al., 2017). Furthermore, the production of phenylamide
phytoalexins, triticamides A–C, was also induced in pathogen-infected
tissues (Ube et al., 2019a). In the present study, metabolic changes in
barley leaves that received exogenous stimuli were analyzed, and two
inducible chalcones with antifungal activity were identified. Addition-
ally, JA and OPDA were shown to induce chalcone accumulation in
barley leaves. These findings indicate that chalcone phytoalexin accu-
mulation plays a role in the inducible defensive responses of barley, and
that the response is regulated by oxylipins such as JA and OPDA. The
discovery of this new phytoalexin class in barley emphasizes the mul-
tiplicity of its chemical defense responses.
The molecular formula of 3 was found to be C19H20O6, as determined
by HRMS (m/z 345.1331 [M+H]+), which showed that the methyl
2

N. Ube et al.
Phytochemistry 184 (2021) 112650
Fig. 1. Comparison of total ion and selected ion chromatograms of extracts from CuCl2-treated and control barley leaves.
The barley leaves were treated with 0.5
mM CuCl2 solution by the floating method. Metabolites in the leaves were extracted 72 h after treatment and analyzed by LC-MS. Three independent samples (3
plant/sample) from either CuCl2 treated or control leaves were analyzed, and increased accumulation of 1–3 in the CuCl2 treated leaves was detected in all analyses.
indicated that the tetrasubstituted benzene ring of 3 was asymmetric
Table 1
and the methoxy group that was replaced by a hydroxy group was
1H NMR spectral data (600 MHz) for 1 and 3 in CDCl3.
located at the C-2 position. Indeed, the 1H NMR spectrum of 3 was
Compound 3
identical to that of 2ʹ-hydroxy-3,4,4ʹ,6ʹ-tetramethoxychalcone (TMC,
Compound 1
Detsi et al., 2009). Thus, the identity of 3 was determined and the
Position
1H multi, J (Hz)
Position
1H multi, J (Hz)
structure has been shown in Fig. 2B.
To confirm the structures of 1 and 3, these compounds were syn-
thesized by the condensation reaction of 3,4-dimethoxybenzaldehyde
with 2,4,6-trimethoxy- and 2-hydroxy-4,6-dimethoxy-acetophenones
to give 1 and 3, respectively (Chu et al., 2004), with corresponding
yields of 98% and 31.4%, respectively. The 1H NMR spectra of the
synthesized compounds were identical to those of 1 and 3, thereby
confirming their identities.
H-3, 5
H-7
6.17 (2H, s)
H-3
6.12 (1H, d, 2.4)
5.97 (1H, d, 2.4)
7.80 (1H, d, 15.6)
7.77 (1H, d, 15.6)
7.13 (1H, d, 1.8)
6.90 (1H, d, 8.4)
7.22 (1H, dd, 8.4, 1.8)
3.94 (3H, s)
6.84 (1H, d, 16.2)
7.28 (1H, d, 16.2)
7.06 (1H, d, 1.8)
6.85 (1H, d, 8.4)
7.09 (1H, dd, 8.4, 1.8)
3.91 (3H, s)
H-5
H-8
H-7
H-2′
H-8
H-5′
H-2′
H-5′
H-6′
MeO x 4
H-6′
MeO x 5
3.90 (3H, s)
3.86 (3H, s)
3.93 (3H, s)
3.77 (6H, s)
3.92 (3H, s)
2.3. Identification of 2
3.84 (3H, s)
OH
14.4 (1H, brs)
Compound 2 was purified by preparative HPLC and subjected to LC-
MS analysis. The analysis indicated that the molecular weight of 2 was
292 based on the presence of the [M-H]- ion at m/z 291. LC-MS/MS
analysis of 2 showed fragment ions at m/z 273, 247, and 165 in the
product ion scan mode (Fig. S4). We speculated that 2 might be OPDA
based on the molecular weight of 292 and the detection of [M-H-CO2]-
ion at m/z 247. The accumulation of OPDA was previously demonstrated
in barley leaves in response to infection by Fusarium graminearum Schw.,
the causal agent of Fusarium head blight, and to osmotic stress caused by
group present in 1 was lost in 3. As such, the 1H NMR spectrum of 3 was
similar to that of 1, except for a methoxy signal that was replaced with a
hydroxy proton signal (Table 1, Fig. S3). The four remaining methoxy
groups of 3 gave four signals with different chemical shifts, indicating
asymmetrical substitution of both benzene rings. Furthermore, the tet-
rasubstituted benzene ring protons were observed as two signals at 6.12
and 5.97 ppm. These differences in the 1H NMR spectra of 1 and 3
3

N. Ube et al.
Phytochemistry 184 (2021) 112650
Fig. 2. 1H–1H COSY correlations (indicated by blue bold line) of 1 (A) and chemical structures of compounds 1–3 (B). (For interpretation of the references to color in
this figure legend, the reader is referred to the Web version of this article.)
sorbitol (Chamarthi et al., 2014; Kramell et al., 2000). The spectra and
retention time of 2, which were determined by LC-MS and LC-MS/MS
analysis, were compared with those of authentic OPDA, which
confirmed that this compound was the identity of 2.
did not change in the intact and control leaves although a marginal in-
crease in the amount of 3 was detected in the control leaves at 72 h.
Compound 2 showed a slight increase at 6–12 h in the control leaves.
Additionally, we investigated the accumulation of other oxylipins,
JA, JA-Ile, 9-KODE, and 13-KODE because the accumulation of these
oxylipins was observed in response to pathogen infection in other plants
(Wakuta et al., 2011; Vollenweider et al., 2000; Horie et al., 2015;
Nishiguchi et al., 2018). The amounts of these oxylipins also increased
after inoculation with B. sorokiniana, where JA and JA-Ile started
increasing 6 h after inoculation and reached their maxima at 12 h,
whereas 9- and 13-KODEs gradually increased throughout the experi-
mental period up to 72 h after inoculation (Fig. 3D–G).
2.4. Accumulation of methoxychalcones and oxylipins after pathogen
infection in barley leaves
The kinetics of the accumulation of PMC (1, Fig. 3A), TMC (3,
Fig. 3B), and OPDA (2, Fig. 3C) were investigated in B. sorokiniana-
infected leaves. The amounts of 1 and 3 gradually increased 24 h after
inoculation, showing a monotonically increasing trend up to 72 h. On
the other hand, 2 showed different kinetics, wherein after the inocula-
tion, the amount of 2 began to increase at an earlier point than those of
PMC and TMC, and reached a maximum 12 h later. The amounts of 1–3
Fig. 3. The kinetics of the accumulation of 2ʹ,3,4,4ʹ,6ʹ-pentamethoxychalcone (PMC, A), 2ʹ-hydroxy-3,4,4ʹ,6ʹ-tetramethoxychalcone (TMC, B), 12-oxo-phytodienoic
acid (OPDA, C), jasmonic acid (JA, D), JA-Ile (E), 9-oxooctadeca-10,12-dienoic acid (9-KODE, F), and 13-oxooctadeca-9,11-dienoic acid (13-KODE, G) in
B. sorokiniana-infected leaves. The metabolites in the leaves were extracted at 0, 6, 12, 24, 48, and 72 h after inoculation.
Data represent mean and standard deviation values obtained from three independent samples (3 plant/sample). Asterisks indicate a statistically significant difference
from the values of the intact leaves (*p < 0.05; **p < 0.01; Dunnett’s test).
4

N. Ube et al.
Phytochemistry 184 (2021) 112650
2.5. Accumulation of 1–3 in leaves treated with CuCl2 and oxylipins
H. bogdanii Wil., H. brachyantherum Nevski. ssp. californicum (Cov. &
Steb.) Both. & et al., H. chilense Roem. & Schult., H. flexuosum Steud.,
H. pusillum Nutt., and H. jubatum L. in the I clade. We also included
wheat and rice in the analysis. However, no accumulation of 1 or 3 was
detected in any of the analyzed species, suggesting that the accumula-
tion of methoxylchalcones was specific to cultivated barley (data not
shown).
We treated the leaf segments with 0.5 mM CuCl2 solution for 72 h
and the amounts of 1–3 in the treated leaves were found to be 4.8-, 58.8-
, 14.8, -fold higher, respectively, than those in control leaves (Fig. 4A).
To examine the effects of oxylipin treatment on the accumulation of
1–3, droplets of 1 mM JA, OPDA, and 9-KODE solutions were placed on
the third leaves. Their concentrations were considered on the basis of
the effective concentrations for the induction of metabolic changes in
barley and rice (Ishihara et al., 2002; Nishiguchi et al., 2018). Treatment
with JA and OPDA elicited the accumulation of 1 and 3 (Fig. 4B). The
respective concentrations of 1 and 3 in JA-treated leaves were 25.5 and
15.3 nmol/g FW, and their concentrations in OPDA-treated leaves were
23.4 and 10.6 nmol/g FW, respectively. In contrast, 9-KODE treatment
did not significantly affect the accumulation of PMC or TMC. We also
included triticamides A–C in the analysis because they have been
denoted as phytoalexins of barley (Ube et al., 2019b). However, triti-
camides were not detected in JA-treated leaves after treatment with any
oxylipins.
3. Discussion
To date, 2ʹ,3,4,4ʹ,6ʹ-pentamethoxychalcone (PMC) has not been iso-
lated from plants although the other PMC, 2′-hydroxy-3,4,3′,4′,6′-pen-
tamethoxychalcone, has been identified in Citrus species (Iwase et al.,
2001; Li et al., 2006). Presence of 2ʹ-Hydroxy-3,4,4ʹ,6ʹ-tetramethox-
ychalcone (TMC) has been reported in the root bark of Pongamia pinnata
L. (Tanaka et al., 1992) and Bidens parviflora Willd. (Li et al., 2008). We
detected PMC and TMC at concentrations higher than 100 nmol/g FW in
B. sorokiniana-infected leaves (Fig. 3), and these concentrations were in
the range where inhibition of fungal germination was observed (Fig. 6).
Thus, PMC and TMC likely exert antifungal activity as chalcone-type
phytoalexins and function as a part of the chemical barrier against
pathogens.
2.6. Antifungal activities of 1 and 3
The finding of induced accumulation of PMC and TMC expands the
list of phytoalexins in barley. In this study, their accumulation was
observed in the pathogen-infected and CuCl2-treated leaves (Fig. 1). In
addition to methoxylchalcones, triticamides and indole amines were
induced in pathogen infected and CuCl2-treated barley leaves (Ishihara
et al., 2017; Ube et al., 2019a, 2019b). These results indicated that
barley possesses multiplicity of phytoalexin to prevent pathogen
infection.
The antifungal activities of 1 and 3 against B. sorokiniana and
F. graminearum were evaluated in an assay for the inhibition of conidial
germination. Compounds 1 and 3 showed inhibitory effects on the
conidial germination of both fungi, and almost completely inhibited
them at a concentration of 1000 μM (Fig. 5).
2.7. Accumulation of 1 and 3 in barley cultivars, wild Hordeum species,
wheat, and rice
The multiplicity of phytoalexin production has been well known in
rice and maize. Approximately twenty compounds have been identified
as phytoalexins in rice, and they are classified into diterpenoids, flavo-
noids, and phenylamides (Yamane, 2013; Park et al., 2013; Morimoto
et al., 2018). In maize, infection with pathogens induced the accumu-
lation of kauralexins (Schmelz et al., 2011) and zealexins (Huffaker
et al., 2011), which belong to diterpenoids and sesquiterpenoids,
respectively. Phytoalexins with different structural characteristics likely
demonstrate different antimicrobial spectra. Indeed, the rice flavonoid
phytoalexins, sakuranetin and naringenin, showed distinct antimicro-
bial activities against fungal and bacterial pathogens. Sakuranetin
showed strong inhibitory activity against fungal pathogens, while nar-
ingenin exhibited the activity against bacterial pathogens, but exerted
almost no inhibitory effect on fungal pathogens (Murata et al., 2020).
The general accumulation of 1 and 3 in barley cultivars was inves-
tigated using seven cultivars: ‘Shunrei’, ‘Morex’, ‘Bowman’, ‘Steptoe’,
‘Yumesakiboshi’, ‘CDC-Fibar’, and ‘Sachiho-Golden’. As JA is a defen-
sive hormone and induces metabolic changes in various plant species,
we used 0.5 M solution of JA as an elicitor. The increased accumulation
of both 1 and 3 in the JA-treated leaves was detected in all cultivars, and
their amounts were 21.2–71.3 nmol/g FW for PMC and 6.7–36.0 nmol/g
FW for TMC, indicating that these compounds were ubiquitously present
in barley cultivars (Fig. 6).
We then expanded the analysis to 10 wild Hordeum species: H. vulgare
L. ssp. spontaneum (C. Koch.) in the H clade; H. murinum subsp. glaucum
(Steudel.) Tzvelev. and H. murinum subsp. leporinum (Link.) in the Xu
clade; H. marinum subsp. marinum Hudson. in the Xa clade, and
Fig. 4. Accumulation of TMC, PMC, and OPDA in barley leaves treated with 0.5 mM CuCl2 (A) and oxylipins (B)
For the treatment of CuCl2, the third leaves of barley seedlings were floated in a 0.5 mM CuCl2 solution containing 0.25% Tween20, and in distilled water containing
0.25% Tween20 as a control for 72 h. For treatments with oxylipins, droplets of 1 mM solutions of JA, OPDA, and 9-KODE containing 0.25% Tween20 were placed on
leaves and incubated for 72 h. Droplets of distilled water containing 0.25% Tween20 were placed on leaves as a control. Data represent mean and standard deviation
values obtained from three independent samples (3 plant/sample). Asterisks indicate a statistically significant difference from the value of the control (*p < 0.05; **p
< 0.01; Student’s t-test).
5

N. Ube et al.
Phytochemistry 184 (2021) 112650
Fig. 5. Antifungal activities of PMC and TMC.
The images show the appearance of the conidia of F. graminearum (Fg) and B. sorokiniana (Bs) when treated with distilled water as a control and with 1000
μ
М PMC
and TMC solutions (A). The conidia of Fg and Bs were incubated with PMC (B) and TMC (C) at various concentrations between 0 and 1000
μM. Scale bar represents
50 μm (A). Germination rates in the PMC and TMC solutions were determined after a 6-h incubation for Fg and after an 8-h incubation for Bs. Data represent mean
and standard deviation values obtained from three independent samples. Letters (a–e) represent significant differences at p < 0.05, as measured using the Tukey-
Kramer test.
species, or the wild relative of cultivated barley, H. vulgare ssp. sponta-
neum. Thus, the biosynthesis of methoxychalcones may have been ac-
quired during the domestication process of barley. However, barley has
been suggested to have been domesticated from H. vulgare ssp. sponta-
neum at least twice, and there is presumably a considerable natural
variation in the specialized metabolism of H. vulgare subsp. spontaneum
(Pourkheirandish et al., 2015). Analysis of methoxychalcones in various
strains of H. vulgare subsp. spontaneum is needed to address the evolu-
tionary origin of their biosynthetic pathway.
The expression of the chalcone synthase gene HvCHS2 (accession no.
Q96562) was upregulated in powdery mildew-infected and UV-treated
barley leaves (Christensen et al., 1998a), although inducible flavo-
noids themselves have not been identified. HvCHS2 showed a preference
for caffeoyl- and feruloyl-CoAs as substrates over coumaroyl- and
cinnamoyl-CoAs. Thus, if this enzyme is involved in the biosynthesis of
methoxychalcones, 2ʹ,3,4,4ʹ,6ʹ-pentahydroxychalcone or 2ʹ,4,4ʹ,6ʹ-tet-
Fig. 6. Accumulation of PMC and TMC in JA-treated leaves of various barley
cultivars. Leaf segments were treated with 0.5 M JA solution for 72 h by floating
method. Distilled water containing 0.25% Tween20 was used as a control. Data
represent mean and standard deviation values obtained from three independent
samples (3 plant/sample). Asterisks indicate a statistically significant difference
from the value of the control (*p < 0.05; **p < 0.01; Student’s t-test).
rahydroxy-3-methoxychalcone—which are formed from caffeoyl- and
feruloyl-CoAs, respectively—are the first intermediates in their biosyn-
thetic pathway.
Two methyltransferases that accept flavonoids as substates have
been characterized in barley. Christensen et al. (1998b) reported the
expression of a flavonoid 7-O-methyltransferase gene (OMT, accession
no. X77467.1) was upregulated in barley leaves in response to powdery
mildew infection. Furthermore, when this OMT-favored apigenin served
as a substrate, genkwanin (7-O-methylapigenin) was generated. Zhou
et al. (2008) characterized the barley flavone methyltransferase-like
gene (HvOMT1, accession no. BI956358). HvOMT1 preferred tricetin
(5,7,3ʹ,4ʹ,5ʹ-pentahydroxyflavone) as a substrate and generated its 3ʹ,
5ʹ-dimethyl derivative, tricin, as the major product. These flavone
O-methyltransferases demonstrated clear regiospecificity in terms of
They are structurally different only in the substituent (methoxyl or hy-
droxyl group) at the 7-position of the flavanone skeleton. It is thus
reasonable to assume that the production of multiple phytoalexins is a
strategy for plants to counter
microorganisms.
a
wide array of pathogenic
The distribution of PMC and TMC was limited in cultivated barley,
and these compounds were not detected in wheat, rice, other Hordeum
6

N. Ube et al.
Phytochemistry 184 (2021) 112650
methylation position. It is probable that multiple methyltransferases
with different substrate- and regio-specificities are involved in the
biosynthesis of PMC and TMC to ensure completion of the methylation
reactions. Feeding experiments using labeled precursors as well as an-
alyses of the substrate- and regio-specificities of inducible methyl-
transferases and chalcone synthases, are needed to elucidate the precise
methylation order in the biosynthesis of TMC and PMC.
5. Experimental
5.1. Instrumentation
1H and 2D (1H–1H COSY) NMR spectra were recorded using the
Avance II instrument (Bruker, Billerica, MA, USA). HR-MS was
measured using the Exactive mass spectrometer (Thermo Fisher Scien-
tific, Waltham, MA, USA). ESI-MS measurements were performed using
the Quattro Micro API mass spectrometer (Waters, Milford, MA, USA)
connected to the Acquity UPLC system (Waters). HPLC was performed
using the 10 A HPLC system (Shimadzu, Kyoto, Japan).
Induced accumulation of OPDA, JA, and JA-Ile in response to
exogenous stimuli, such as osmotic stress and pathogen infection, has
been demonstrated in barley (Kramell et al., 2000; Kumaraswamy et al.,
2011). In addition to these compounds, we found that 9- and 13-KODEs
accumulated in pathogen-infected barley leaves (Fig. 4). However,
OPDA, JA, and JA-Ile accumulated earlier than the 9- and 13-KODEs.
Furthermore, treatments of barley leaves with JA and OPDA induced
PMC and TMC accumulation, but treatment with 9-KODE did not. This
indicated that these oxylipins play different roles at different times
during the disease response in barley.
5.2. Plant materials and pathogenic fungi
Barley (Hordeum vulgare L. ‘Shunrei’ (Poaceae)] and wheat [Triticum
aestivum L. ‘Norin 61’ (Poaceae)] were purchased from JA Inaba (Tot-
tori, Japan) and Tsurushin Shubyo (Kyoto, Japan), respectively. Seeds of
rice [Oryza sativa L. ‘Nipponbare’ (Poaceae)] were acquired from the
stock of the Faculty of Agriculture at Tottori University (Tottori, Japan).
The seeds of these species were immersed in distilled water for one
night, sowed on a 1:1 (v/v) mixture of vermiculite (Shoei Sangyo,
Okayama, Japan) and culture soil (Iris Ohyama, Sendai, Japan), and
incubated at 25 ◦C with 14-h light/10-h dark cycles for 3 weeks.
H. vulgare L. subsp. spontaneum (C. Koch.) Thell., H. murinum subsp.
glaucum (Steudel.) Tzvelev., H. marinum subsp. marinum Hudson, H.
chilense Roem. & Schult., H. brachyantherum Nevski. subsp. californicum
(Cov. & Steb.) Both. & et al., H. bogdanii Wil., H. flexuosum Steud., H.
pusillum Nutt. (these nine species are all diploids), H. murinum subsp.
leporinum (Link.) Arcang., and H. jubatum L. (both species are tetra-
ploids) were obtained from the stocks of the Institute of Plant Science
and Resources, Okayama University (Kurashiki, Okayama, Japan). The
taxonomy of the Hordeum species is in accordance with the description
reported by von Bothmer et al. (1995).
JA and OPDA are signal molecules involved in stress responses and
trigger the accumulation of defensive metabolites in plants (Schmelz
et al., 2011; Huffaker et al., 2011; Murata et al., 2020; Ube et al., 2019a).
In barley, PMC and TMC were induced in the JA-treated leaves, whereas
triticamides were not induced by JA treatment. Ishihara et al. (2002)
reported that 6ʹʹʹ-feruloylsaponarin and 6ʹʹʹ-sinapoylsaponarin accumu-
lated in JA-treated leaves, but not in leaves treated with other plant
hormones in barley. The signaling role of JA in promoting sakuranetin
production was clearly demonstrated by rice mutants lacking the
JA-signaling pathway, wherein sakuranetin did not accumulate in
response to pathogen attack (Ogawa et al., 2017). In Petroselinum
crispum (Mill.) fuss cell culture, the accumulation of flavonoid phyto-
alexin apiin was induced by OPDA treatment (Dittrich et al., 1992).
These findings suggest that the JA-mediated signaling pathway is closely
associated with the activation of the flavonoid biosynthetic pathway.
The functions of KODEs remain unclear, although their accumulation
in several plant species has been reported (Nishiguchi et al., 2018;
Vollenweider et al., 2000; Feng et al., 2007; Gao et al., 2007). In rice, the
treatment of leaves with KODEs caused the accumulation of defensive
metabolites sakuranetin and serotonin (Nishiguchi et al., 2018). In
Arabidopsis thaliana, it has been shown that infection of Pseudomonas
syringae pv. tomato Van Hall led to high concentrations of KODE accu-
mulation in the leaves (Vollenweider et al., 2000). The exogenous
application of KODEs to Arabidopsis leaves resulted in cell death and the
expression of the glutathione S-transferase gene (GST1). GST1 expres-
sion is known to occur in pathogenesis and is caused by oxidative stress
(Jabs et al., 1996; Mauch and Dudler, 1993). Therefore, KODE accu-
mulation was considered to be intimately related to the expression of
stress responses. The accumulation of KODEs may be a general response
of plants towards a pathogen attack; however, the function of KODEs in
barley requires elucidation.
B. sorokiniana (Sacc. ex Sorok.) Shoem. (OB-25-1), the causal agent
of spot blotch of the poaceous species, was a stock from the Laboratory
of Natural Product Chemistry, Faculty of Agriculture, Tottori University.
The causal agent of Fusarium head blight, F. graminearum Schw. (H-3),
was a stock from the National Agriculture and Food Research Organi-
zation. B. sorokiniana and F. graminearum were inoculated on V8 agar
plates and cultured for 1 week and 2 weeks, respectively, at 25 ◦C under
a black light bulb (BLB: FL15BL-B, Hitachi, Tokyo) to obtain conidia.
5.3. Chemicals
JA was prepared by alkaline hydrolysis of methyl jasmonate (Tokyo
Chemical Industry, Tokyo, Japan). JA-Ile, 9-KODE, and 13-KODE were
purchased from Funakoshi (Tokyo). OPDA was purified from injured
barley leaves. Aerial parts of barley leaves (213 g) were ground in liquid
N2 with a mortar and pestle. The ground residue was incubated at 30 ◦C
for 6 h and immersed in 2.1 L of 80% methanol for 24 h. The extract was
evaporated in vacuo and partitioned with water and EtOAc. The EtOAc
layer was evaporated to dryness and dissolved in 50% methanol. This
solution was subjected to Sep-Pak Plus C18 environmental (Waters) and
eluted with 50%, 60%, 70%, 80%, 90%, and then 100% methanol in
water. These fractions were subjected to LC-MS analysis to monitor the
elution of OPDA. The fractions containing OPDA (60% and 70% meth-
anol fractions) were combined, concentrated, and subjected to prepar-
ative HPLC. The HPLC conditions were as follows: column, Cosmosil
5C18-AR-II (Nacalai Tesque, Kyoto), 20 I.D. × 250 mm; solvents, 0.1%
formic acid water (A) and methanol (B); elution, 75% isocratic B/(A +
B); flow rate, 7.0 mL/min; detection, 220 nm; column temperature,
40 ◦C. OPDA was eluted at 26.2 min, and the fraction corresponding to it
was collected and concentrated to dryness to yield OPDA (1.7 mg). The
identity of the compound was confirmed by comparing the 1H NMR
spectra with reported data (Nonaka et al., 2010). The preparation of
triticamides A–C was conducted as described in a previous study (Ube
4. Conclusion
PMC (1), OPDA (2), and TMC (3) accumulation in barley was
induced in response to pathogen attack. Based on PMC and TMC anti-
fungal activity, we proposed that these two methoxychalcones serve as
chalcone-type phytoalexins. Since indole amine derivatives (Ishihara
et al., 2017) and triticamides A–C (Ube et al., 2019a,b) have been pre-
viously reported as phytoalexins, barley plants utilize multiple classes of
phytoalexin as a chemical defense barrier. Treatments of barley leaves
with JA and OPDA promoted the accumulation of PMC and TMC, sug-
gesting that chalcone phytoalexin biosynthesis was regulated by
oxylipins.
7

N. Ube et al.
Phytochemistry 184 (2021) 112650
et al. 2019a, 2019b).
external standards to determine the contents of compounds.
5.4. Treatment of barley leaves with chemicals
5.7. Isolation of compounds 1 and 3
We used the following two methods to treat leaves with compounds:
floating method and droplet method. In the floating method, leaf seg-
ments were floated in a solution of compounds containing 0.25%
Tween20 and incubated for 72 h at 25 ◦C with 14-h light/10-h dark
Aerial parts (485 g) of barley seedlings were immersed in a 0.1 mM
CuCl2 solution and incubated at 23 ◦C with 14-h light/10-h dark cycles.
After a 72-h incubation, metabolites in the aerial parts were extracted
with 80% methanol for 24 h, and the obtained extract was evaporated in
vacuo. The residue was partitioned with water and EtOAc, and the EtOAc
layer was evaporated to dryness. The EtOAc extract was subjected to
column chromatography using silica gel (Daisogel IR-60-63/210, Osaka
Soda, Osaka, Japan). The column was eluted with a mixture of acetone
and hexane. The concentration of acetone was increased from 0% to
100% in increments of 10%. Compounds 1 and 3 were detected in the
30% acetone fraction, which was then subjected to ODS column chro-
matography (Cosmosil 75C18-PREP, Nacalai Tesque). The column was
eluted with 50%, 60%, 70%, and 80% methanol in water (150 mL each).
Compounds 1 and 3 were eluted in 60% and 70% methanol fractions.
These fractions were combined, evaporated to a small volume, and
subjected to preparative HPLC under the following conditions: column,
Cosmosil 5C18-AR-II (Nacalai Tesque), 10 I.D. × 250 mm; solvents,
water (A) and acetonitrile (B); elution, 60% B/(A + B); flow rate, 3.0
mL/min; detection, 280 nm; column temperature, 40 ◦C. Compounds 1
and 3 were eluted at 14.2 and 17.8 min, respectively.
cycles. In the droplet method, droplets (5 μL) of a solution of compounds
containing 0.25% Tween20 were placed on a barley leaf at 1.0-cm in-
tervals. The seedlings were incubated for 72 h at 25 ◦C with 14-h light/
10-h dark cycles.
5.5. Inoculation of pathogens
The conidia of B. sorokiniana were inoculated to the third leaves of 3-
week-old barley seedlings. Droplets (5 μL) of the conidial suspension (5
× 105 conidia/mL in distilled water containing 0.25% Tween 20) were
placed on a barley leaf at 1.0-cm intervals. Droplets of distilled water
containing 0.25% Tween 20 were also placed on the leaves as a control.
Intact leaves without any treatments were also extracted. The inoculated
seedlings were kept in a moist, air-tight bag for 24 h, and then removed
from the bag and further incubated at 25 ◦C with 14-h light/10-h dark
cycles. The metabolites in the leaves were extracted at 0, 6, 12, 24, 48,
and 72 h after inoculation.
Compound 1 (2ʹ,3,4,4ʹ,6ʹ-pentamethoxychalcone), 1.2 mg; HR-MS
(positive ESI): m/z 359.1487 [M+H]+ (calcd. for C20H23O6, m/z
359.1494); UV–Vis (acetonitrile-water containing 0.1% formic acid):
5.6. Analyses of oxylipins and methoxylchalcones
λ
max 223 and 340 nm; 1H NMR data are shown in Table 1.
Compound 3 (2ʹ-hydroxy-3,4,4ʹ,6ʹ-tetramethoxychalcone), 1.8 mg;
In all analyses, we extracted metabolites in the leaves by immersing
the leaves in 80% methanol for 24 h. The volume of 80% methanol was
fixed at a ratio of 10 mL/g FW leaves. The concentrations of metabolites
in the leaves were calculated under the assumption that the concentra-
tions in the leaves were 10 times higher than those in the extracts.
For the detection of metabolites induced by CuCl2 treatment, the
extracts of CuCl2-treated leaves were analyzed by LC-MS (Quattro Micro
API mass spectrometer connected with Aquity UPLC, Waters). The LC
conditions were as follows: column, Acquity UPLC BEH C18 (Waters),
HR-MS (positive ESI): m/z 345.1331 [M+H]+ (calcd. for C19H21O6, m/z
345.1338); UV–Vis (acetonitrile-water containing 0.1% formic acid):
λ
max 222 and 375 nm; 1H NMR data are shown in Table 1.
5.8. Syntheses of 1 and 3
Compound 1 was synthesized according to the methods described by
Chu et al. (2004). A mixture of 2,4,6-trimethoxyacetophenone (210 mg,
1.0 mmol) and 3,4-dimethoxybenzaldehyde (191 mg, 1.15 mmol) in 20
mL of anhydrous EtOH was stirred at room temperature for 20 min. A
solution of 50% KOH (40 mL) was added to the mixture and the mixture
was stirred at room temperature for 3 h. The resulting solution was
neutralized by the addition of 4 M HCl and extracted with CH2Cl2. The
organic layer was dried over Na2SO4, filtered, and the solvent was
removed under reduced pressure. The residue was subjected to silica gel
column chromatography (Daisogel IR-60-63-210). The compounds were
eluted with EtOAc/n-hexane (3:7, v/v), and the fraction volume was set
to 5 mL. The fractions containing 1 were combined and concentrated to
give the product (353 mg, 99% yield). 1H NMR (600 MHz, CDCl3) δH:
3.77 (6H, s, OMe × 2), 3.86, 3.90, and 3.91 (3H each, s, OMe × 3), 6.17
(2H, s, H–3,5), 6.84 (1H, d, J = 16.0 Hz, H-7), 6.86 (1H, d, J = 8.0 Hz,
H-5′), 7.07 (1H, s, H-2′), 7.09 (1H, d, J = 8.0 Hz, H-6′), and 7.29 (1H, d,
J = 16.0 Hz, H-8).
2.1 mm × 50 mm (1.7 μm); flow rate, 0.2 mL/min; column temperature,
40 ◦C; solvents, 0.1% formic acid in water (A) and 0.1% formic acid in
acetonitrile (B); gradient, 5%–100% A/(A + B) within 10 min. The MS
scan conditions were as follows: capillary voltage, 3 kV; cone voltage,
20 V; source temperature, 120 ◦C; desolvation temperature, 350 ◦C;
desolvation gas flow, 600 L/h; cone gas flow, 50 L/h; m/z range,
150–1000.
Compounds 1–3 and KODEs were analyzed using LC-MS/MS
(Quattro Micro API mass spectrometer connected with Aquity UPLC,
Waters) coupled with multiple reaction monitoring (MRM) methods.
The LC conditions were as follows: column, Acquity UPLC BEH C18
(Waters), 2.1 mm × 50 mm (1.7 μm); flow rate, 0.2 mL/min; column
temperature, 40 ◦C; solvents, 0.1% formic acid in water (A) and 0.1%
formic acid in acetonitrile (B); gradient for OPDA, 9-KODE, and 13-
KODE, 40%–80% A/(A + B) within 5 min; gradient for PMC and TMC,
5%–70% A/(A + B) within 10 min. The MRM conditions were optimized
using authentic compounds (Table S1). The amounts of compounds were
determined using external standards.
Compound 3 was synthesized from 2-hydroxy-4,6-dimethoxyaceto-
phenone (98 mg, 0.5 mmol) and 3,4-dimethoxybenzaldehyde (96 mg,
0.58 mmol) using the same method utilized for the synthesis of 1. After
extraction with CH2Cl2, the residue of 3 was recrystallized from EtOH
and purified 3 was obtained (54.0 mg, 31% yield). 1H NMR (600 MHz,
CDCl3) δH: 3.83, 3.90, 3.92, 3.93 (3H each, s, OMe × 4), 5.95 (1H, d, J =
1.8 Hz, H-5), 6.10 (1H, d, J = 1.8 Hz, H-3), 6.89 (1H, d, J = 8.0 Hz, H-5′),
7.21 (1H, d, J = 8.0 Hz, H-6′), 7.25 (1H, s, H-2′), 7.75 (1H, d, J = 15.5
Hz, H-8), 7.79 (1H, d, J = 15.5 Hz, H-7), and 14.40 (1H, bs, –OH).
The amounts of JA and JA-Ile were determined by LC-MS/MS anal-
ysis in the MRM mode with the Shimadzu LCMS-8040 system (Shi-
madzu). The HPLC conditions were as follows: column, Inertsil ODS-4 5
μ
m (GL Sciences, Tokyo, Japan), 4.6 mm × 150 mm; column tempera-
ture, 40 ◦C; solvent, water (A) and acetonitrile (B); gradient, 10% B/(A
+ B) (0–2 min), 10%–100% (2–12 min), 100% (12–13 min); flow rate,
0.4 mL/min. ESI-MS/MS analysis in the MRM mode was performed in
the negative mode. For quantitative analysis of these compounds, ESI-
MS/MS analysis in the MRM mode was performed in the negative
mode for JA and JA-Ile, and the MRM conditions were optimized using
authentic compounds (Table S2). Authentic JA and JA-Ile were used as
5.9. Antifungal activity
The antifungal activities of PMC and TMC for B. sorokiniana and
F. graminearum were evaluated as described by Ube et al. (2019a). The
8

N. Ube et al.
Phytochemistry 184 (2021) 112650
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Declaration of competing interest
Ishihara, A., Kumeda, R., Hayashi, N., Yagi, Y., Sakaguchi, N., Kokubo, Y., Ube, N.,
Tebayashi, S., Ueno, K., 2017. Induced accumulation of tyramine, serotonin, and
related amines in response to Bipolaris sorokiniana infection in barley. Biosci.
Biotechnol. Biochem. 81, 1090–1098. https://doi.org/10.1080/
The authors declare no conflicts of interest associated with this
manuscript.
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Acknowledgments
This work was supported by the Japan Society of the Promotion of
the Science (JSPS) KAKENHI (Grant Number 18J11919 to N.U.) and by
the Ministry of Education, Culture, Sports, Science, and Technology
(MEXT) as part of a joint research program implemented at the Institute
of Plant Science and Resources, Okayama University, Japan, and the
Arid Land Research Center, Tottori University, Japan.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.phytochem.2020.112650.
Kumaraswamy, G.K., Bollina, V., Kushalappa, A.C., Choo, T.M., Dion, Y., Rioux, S.,
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