International Journal of

Molecular Sciences

Article
Synergistic Effect of Methyl Jasmonate and Abscisic Acid
Co-Treatment on Avenanthramide Production in
Germinating Oats
Soyoung Kim 1,2,† , Tae Hee Kim 1,3,† , Yu Jeong Jeong 1 , Su Hyun Park 1,2 , Sung Chul Park 1 , Jiyoung Lee 1 ,
Kwang Yeol Yang 3 , Jae Cheol Jeong 1, * and Cha Young Kim 1, *

1 Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB),
Jeongeup 56212, Korea; cherish767@kribb.re.kr (S.K.); xogml5344@naver.com (T.H.K.);
yjjeong@kribb.re.kr (Y.J.J.); suhyun@kribb.re.kr (S.H.P.); heypsc@kribb.re.kr (S.C.P.);
jiyoung1@kribb.re.kr (J.L.)
2 Department of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University,
Gwangju 61186, Korea
3 Department of Applied Biology, College of Agriculture and Life Science, Chonnam National University,
Gwangju 61186, Korea; kyyang@jnu.ac.kr
* Correspondence: jcjeong@kribb.re.kr (J.C.J.); kimcy@kribb.re.kr (C.Y.K.); Tel.: +82-63-570-5001 (C.Y.K.);
Fax: +82-63-570-5009 (C.Y.K.)
† These authors contributed equally to this work.

Abstract: The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful



anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant,


anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with
Citation: Kim, S.; Kim, T.H.; Jeong, methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold
Y.J.; Park, S.H.; Park, S.C.; Lee, J.;
(642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls
Yang, K.Y.; Jeong, J.C.; Kim, C.Y.
(232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide
Synergistic Effect of Methyl
production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment
Jasmonate and Abscisic Acid
increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis
Co-Treatment on Avenanthramide
Production in Germinating Oats. Int.
pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase
J. Mol. Sci. 2021, 22, 4779. https:// (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA
doi.org/10.3390/ijms22094779 treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of
all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression
Academic Editor: Maurizio Battino following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in
avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These
Received: 13 April 2021 findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable
Accepted: 27 April 2021 method for avenanthramide production in germinating oats.
Published: 30 April 2021

Keywords: ABA; Avena sativa; elicitation; MeJA; metabolite farming

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with regard to jurisdictional claims in
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iations.
1. Introduction
Plants are complex organisms that form an important part of our daily lives. In
addition to primary metabolites essential for growth and development, plants produce a
vast number of compounds that play crucial roles in defense and environmental adaptation
Copyright: © 2021 by the authors.
(i.e., secondary metabolites) [1–4]. Secondary metabolites, such as terpenes, alkaloids,
Licensee MDPI, Basel, Switzerland.
flavonoids, and carotenoids, have many industrial uses, including as pharmaceuticals,
This article is an open access article
pesticides, flavorings, and food additives [2,3]. Oats produce a group of phenolic secondary
distributed under the terms and
conditions of the Creative Commons
metabolites known as avenanthramides, which are low-molecular-weight polyphenols
Attribution (CC BY) license (https://
produced only in oats [5] and act as phytoalexins produced in oat leaves in response to
creativecommons.org/licenses/by/ pathogen infection [6] or treatment with elicitors [7–9].
4.0/).

Int. J. Mol. Sci. 2021, 22, 4779. https://doi.org/10.3390/ijms22094779 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 4779 2 of 15

Avenanthramides are endowed with beneficial health properties and possess an-
tioxidant, antipruritic, anti-proliferative, anticancer, and anti-inflammatory effects [10–15].
Moreover, avenanthramides exhibit strong antioxidant properties with up to 30-fold greater
activity than other phenolic compounds produced in oats [16–18]. To date, more than 25
different types of avenanthramides have been identified in oats [19–21]. Avenanthramides
are a group of alkaloids that comprise an anthranilic acid derivative linked to a hydroxycin-
namic acid derivative by an amide bond [5,19,22]. Three major avenanthramide isoforms
[avenanthramide (Avn) A, B, and C] have been identified so far [10], all of which con-
tain 5-hydroxyanthranilic acid and various hydroxycinnamic acids (p-coumaric acid in
Avn A, ferulic acid in Avn B, and caffeic acid in Avn C) [19,23,24]. Avenanthramide
biosynthesis follows the phenylpropanoid pathway (Figure 1), with L-phenylalanine rep-
resenting the starting amino acid for biosynthesis [25]. In the first step, phenylalanine
is transformed into cinnamic acid by phenylalanine ammonia-lyase (PAL) before being
converted into 4-coumaric acid, caffeic acid, and ferulic acid by cinnamate 4-hydroxylase
enzymes [25]. In the next step, 4-coumarate-CoA ligase (4CL) catalyzes the condensation
of 4-coumaroyl-CoA and two malonyl-CoA molecules. The phenylpropanoid pathway
provides precursors for many secondary metabolites in plants, including lignins, antho-
cyanins, and flavonoids [26]. Avenanthramides are synthesized through a condensation
process involving hydroxyanthranilic acid and hydroxycinnamoyl-CoA, which is catalyzed
by the key enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl
transferase (HHT) [7,8,21]. To date, six HHT and one caffeoyl-CoA O-methyltransferase
(CCoAOMT) cDNAs have been isolated from oats [8]. The encoded isoforms of AsHHT1–3
(~95 to 98%) and AsHHT4–6 (~95%) share high amino acid sequence identity, whereas
isoforms of AsHHT4–6 exhibit somewhat low amino acid sequence identity (~82%) with
AsHTT1–3 [21]. A previous study reported that expression of AsHHT1 and CCoAOMT
is concomitantly induced along with phytoalexin accumulation by a vitorin elicitor in
Pc-2/Vb oat lines [8].
Metabolite farming is an agricultural method or technology used to induce the pro-
duction of secondary metabolites through the use of various elicitors in plants, cells, tissues,
and organs [3,27–30]. The aim is to increase the value of plants or plant cell cultures by
enhancing the contents of the main bioactive substances. These methods are applied to
generate useful natural materials in high-value-added foods, cosmetics, and pharmaceuti-
cal products and as a key approach for enhancing the competitiveness of agriculture as an
industry [31].
In this study, the application of a small-scale metabolite-farming system to increase
the avenanthramide content in germinating oats via treatment with various elicitors was
investigated. The results suggested that co-treatment of germinating oats with methyl
jasmonate (MeJA) or abscisic acid (ABA) showed a significant and synergistic effect on the
expression of structural genes involved in avenanthramide biosynthesis, thereby leading
to enhanced production of avenanthramides. This work would provide a basis for applica-
tions of elicitor-mediated metabolite farming to develop high-value-added plants capable
of increasing the accumulation of bioactive metabolites.
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Figure Proposedbiosynthetic
1. Proposed
Figure 1. biosynthetic pathway
pathway of avenanthramides
of avenanthramides in oats.
in oats. The avenanthramide-
The avenanthramide-bio-
biosynthetic
synthetic pathway was proposed based on information from the KEGG databasedatabase
pathway was proposed based on information from the KEGG (http://
(http://www.ge-
www.genome.jp/kegg/kegg2.html;
nome.jp/kegg/kegg2.html; accessed on accessed
25 March on2020).
25 March
PAL,2020). PAL, phenylalanine
phenylalanine ammonia
ammonia lyase; C4H,
lyase; C4H,4-hydroxylase;
cinnamate cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA
4CL, 4-coumarate-CoA ligase; CCoAOMT,
ligase; CCoAOMT, caffeoyl-CoAcaffeoyl-CoA O-
O-methyltrans-
ferase; HHT, hydroxycinnamoyl-CoA:hydroxyanthranilate
methyltransferase; N-hydroxycinnamoyl
HHT, hydroxycinnamoyl-CoA:hydroxyanthranilate transferase;trans-
N-hydroxycinnamoyl
KEGG,KEGG,
ferase; Kyoto Kyoto
Encyclopedia of Genes
Encyclopedia and Genomes.
of Genes and Genomes.

2.
2. Results
Results
2.1.
2.1. Avenanthramide
Avenanthramide Content
Content of
of Germinating
Germinating Oats
Oats
In
In the
thepresent
presentstudy, avenanthramide
study, avenanthramidecontents in ain
contents time-course experiment
a time-course over 5over
experiment days5
after sowing were analyzed. Figure 2A shows the germinating oat phenotype from days 0
days after sowing were analyzed. Figure 2A shows the germinating oat phenotype from
to 5 of culture. Radicles emerged 1 day after sowing, and the sprouts grew to lengths of up
days 0 to 5 of culture. Radicles emerged 1 day after sowing, and the sprouts grew to
to 5 cm in 5 days. The contents of Avn A and Avn C increased 9.8- and 6.9-fold, respectively,
lengths of up to 5 cm in 5 days. The contents of Avn A and Avn C increased 9.8- and 6.9-
during the day 1, and the total avenanthramide content increased 5.6-fold (205.7 mg/kg
fold, respectively, during the day 1, and the total avenanthramide content increased 5.6-fold
FW) relative to that on day 0 (36.5 mg/kg FW). On day 2, the total avenanthramide content
(205.7 mg/kg FW) relative to that on day 0 (36.5 mg/kg FW). On day 2, the total avenan-
had increased 8.5-fold relative to day 0. Moreover, the highest proportions of Avn A
thramide content had increased 8.5-fold relative to day 0. Moreover, the highest proportions
(157.3 mg/kg FW), Avn B (154.2 mg/kg FW), and Avn C (152.4 mg/kg FW) were quantified
of Avn A (157.3 mg/kg FW), Avn B (154.2 mg/kg FW), and Avn C (152.4 mg/kg FW) were
on day 3 (Figure 2B). Therefore, day 2 was selected as the optimal time point for elicitor
quantified on day 3 (Figure 2B). Therefore, day 2 was selected as the optimal time point for
treatment to enhance avenanthramide content during oat germination.
elicitor treatment to enhance avenanthramide content during oat germination.
Int. J.J. Mol.
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Figure 2.
Figure Yields of
2. Yields of avenanthramides
avenanthramidesin inoats
oatsat
atdifferent
differentstages
stagesofofgermination.
germination.(A) (A)Oat
Oatseedlings
seedlingsatat
different
differentdevelopmental
developmentalstages.
stages.Oat
Oatseeds
seedswere
weregerminated
germinated in in
pre-wetted
pre-wettedfilter paper
filter forfor
paper 5 days.
5 days.
(B)
(B) Production
Productionofofavenanthramides
avenanthramides ininoats during
oats duringgermination.
germination.Whole germinating
Whole germinatingoats were har-
oats were
vested on the days indicated after sowing. Twenty germinating oat plants were used for
harvested on the days indicated after sowing. Twenty germinating oat plants were used for avenan- avenan-
thramide quantification at each time point, and the data shown are mean values. Data represent
thramide quantification at each time point, and the data shown are mean values. Data represent
the mean of three independent replicates ± SD. Different letters above the bars indicates significant
the mean of three independent replicates ± SD. Different letters above the bars indicates significant
differences determined by ANOVA followed by a Tukey’s test (p < 0.05).
differences determined by ANOVA followed by a Tukey’s test (p < 0.05).
2.2.
2.2. Induction
Induction of
of Avenanthramide
Avenanthramide Production
Production and
and Expression
Expression of
of Avenanthramide-Biosynthetic
Avenanthramide-Biosynthetic
Genes
Genes inin Germinating
Germinating Oats
Oats Following Treatment with Various Elicitors
Elicitors
Two-day-old
Two-day-old germinating
germinating oats oats were
were treated
treated for for 72
72 h with various
various elicitors,
elicitors, such
such as as
methyl
methyl viologen
viologen (MV; (MV; 10 μM),
µM), MeJA (100 μM), µM), ABA (300 μM), µM), chitosan (Chi; 200 µg/mL),μg/mL),
salicylic acid
salicylic acid(SA;(SA;100 100μM),
µM), andand ethephon
ethephon (ET;(ET;
300300μM), and and
µM), 5-day-old
5-day-old germinating
germinating oats
oats were
were collected
collected and analyzed
and analyzed by high-performance
by high-performance liquidliquid chromatography
chromatography (HPLC) (HPLC)
(Fig-
(Figure
ure 3A).3A).
MeJA MeJAand and
ABAABA effectively
effectively enhanced
enhanced the production
the production of avenanthramides
of avenanthramides in ger-in
germinating
minating oats,oats,
withwithMeJA MeJA treatment
treatment resulting
resulting in increases
in increases of ~2.7-fold
of ~2.7-fold (190.1(190.1
mg/kg mg/kgFW)
FW)
for Avnfor A,
Avn A, 2.5-fold
2.5-fold (260.4(260.4
mg/kg mg/kg
FW) forFW) for B,
Avn Avn B, 2.3-fold
2.3-fold (132.4(132.4
mg/kg mg/kg
FW) forFW) for C,
Avn AvnandC,
and 2.5-fold
2.5-fold (582.9 (582.9
mg/kg mg/kg FW)FW)for for total
total avenanthramides
avenanthramides relative
relative totothe
theethanol
ethanol(EtOH)-
(EtOH)-
treated control
treated control(Avn (AvnA, A,71.1
71.1mg/kg
mg/kg FW;FW;Avn AvnB, B, 103.2
103.2 mg/kg
mg/kg FW;FW;AvnAvn C, 58.3
C, 58.3 mg/kg mg/kgFW;
FW; and total avenanthramides, 232.6 mg/kg FW). Additionally,
and total avenanthramides, 232.6 mg/kg FW). Additionally, ABA-treated seedlings ABA-treated seedlings
showed an
showed an increase
increase in in avenanthramide
avenanthramide production
production of of 2.7-fold
2.7-fold (193.8
(193.8 mg/kg
mg/kg FW) FW) for
for Avn
Avn
A, 2.0-fold (207.4 mg/kg FW) for Avn B, 4.1-fold (241.7 mg/kg FW)
A, 2.0-fold (207.4 mg/kg FW) for Avn B, 4.1-fold (241.7 mg/kg FW) for Avn C, and 2.8-fold for Avn C, and 2.8-fold
(642.9 mg/kg
(642.9 mg/kg FW) FW) forfor total
total avenanthramides
avenanthramidesrelative relativetotothetheEtOH
EtOH control.
control.NoNo other elicitors
other elici-
induced
tors significantly
induced elevated
significantly levelslevels
elevated of avenanthramide
of avenanthramide production relative
production to the to
relative EtOH
the
and distilled water (DW) controls. These results suggested the efficacy
EtOH and distilled water (DW) controls. These results suggested the efficacy of MeJA and of MeJA and ABA
as elicitors
ABA for enhanced
as elicitors for enhancedproduction of avenanthramides
production of avenanthramides in oatinseedlings.
oat seedlings.
The expression levels of genes encoding
The expression levels of genes encoding enzymes (PAL, 4CL,enzymes (PAL, 4CL,CCoAOMT,
CCoAOMT, and and HHT)
HHT)
involved in the avenanthramide-biosynthesis pathway (Figure
involved in the avenanthramide-biosynthesis pathway (Figure 1) were determined by 1) were determined by
quantitative reverse transcription (qRT)-PCR, with the AsActin
quantitative reverse transcription (qRT)-PCR, with the AsActin gene used as an internal gene used as an internal
control. The
control. expressionofofAsPAL,
The expression AsPAL, As4CL,
As4CL,andandAsCCoAOMT
AsCCoAOMT in germinating oats was
in germinating oats signifi-
was
cantly induced by treatments of MeJA and ABA, with 4.2- and 19.7-fold increases in AsPAL,
significantly induced by treatments of MeJA and ABA, with 4.2- and 19.7-fold increases
3.0- and 2.8-fold increases in As4CL, and 2.3- and 1.9-fold increases in AsCCoAOMT, respec-
in AsPAL, 3.0- and 2.8-fold increases in As4CL, and 2.3- and 1.9-fold increases in
tively, relative to the negative control (0.1% EtOH) (Figure 3B,C). The six AsHHT genes
AsCCoAOMT, respectively, relative to the negative control (0.1% EtOH) (Figure 3B,C). The
examined effectively responded to treatments of MeJA and ABA (1.7- to 10.8-fold increases
six AsHHT genes examined effectively responded to treatments of MeJA and ABA (1.7- to
in production). In particular, MeJA treatment increased AsHHT4 expression 10.8-fold, and
10.8-fold increases in production). In particular, MeJA treatment increased AsHHT4
ABA treatment significantly upregulated AsHHT5 expression 6.2-fold in germinating oats.
expression 10.8-fold, and ABA treatment significantly upregulated AsHHT5 expression
Additionally, SA and ET treatments slightly upregulated AsPAL, As4CL, AsCCoAOMT,
6.2-fold in germinating oats. Additionally, SA and ET treatments slightly upregulated
AsHHT1, AsHHT2, AsHHT3, and AsHHT5 expression, whereas those treatments reduced
AsPAL, As4CL, AsCCoAOMT, AsHHT1, AsHHT2, AsHHT3, and AsHHT5 expression,
AsHHT4 and AsHHT6 expression. However, MV and Chi treatments did not significantly
whereas those treatments reduced AsHHT4 and AsHHT6 expression. However, MV and
affect the expression of avenanthramide-biosynthetic genes. These results indicated that
Chi treatments did not significantly affect the expression of avenanthramide-biosynthetic
MeJA and ABA treatments of germinating oats effectively enhanced the expression of the
genes.
six AsHHTThese genesresults indicated
analyzed in thisthat MeJA and ABA treatments of germinating oats
study.
effectively enhanced the expression of the six AsHHT genes analyzed in this study.
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Figure3.
Figure Avenanthramide content
3. Avenanthramide content and
andrelative
relativeexpression
expressionlevels
levelsofofbiosynthetic
biosynthetic genes in in
genes germinating
germi-
nating oats treated
oats treated with various
with various elicitors.
elicitors. Two-day-old
Two-day-old oat seedlings
oat seedlings were were treated
treated usingusing various
various elicitors
elicitors for 3 days. (A) Quantitative analysis of avenanthramide production by
for 3 days. (A) Quantitative analysis of avenanthramide production by HPLC. Ethanol (0.1% EtOH)HPLC. Ethanol
(0.1% EtOH)
was used as awas used control
solvent as a solvent control
for MeJA andfor MeJA
ABA, and
and ABA, and
distilled waterdistilled water
(DW) was (DW)
used as was used
control for
as control for MV, Chi, SA, and ET. (B) The relative expression level of avenanthramide-biosyn-
MV, Chi, SA, and ET. (B) The relative expression level of avenanthramide-biosynthetic genes under
thetic genes under treatment with various elicitors dissolved in ethanol (0.1% EtOH). (C) The rela-
treatment with various elicitors dissolved in ethanol (0.1% EtOH). (C) The relative expression level
tive expression level of avenanthramide-biosynthetic genes under treatment with various elicitors
of avenanthramide-biosynthetic genes under treatment with various elicitors dissolved in distilled
dissolved in distilled water (DW). Data represent the mean of three independent replicates ± SD.
water (DW).
Student’s Data
t-test; < 0.05. the mean of three independent replicates ± SD. Student’s t-test; * p < 0.05.
* prepresent
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2.3.
2.3.Enhanced
EnhancedProduction
ProductionofofAvnenathramides
Avnenathramides by by MeJA
MeJA and/or
and/or ABA
ABA Treatment
Treatment
To
To determine
determine the the optimal
optimal concentration
concentration of of MeJA
MeJA forfor enhancing
enhancing the the total
total avenan-
avenan-
thramide
thramideyield
yieldin ingerminating
germinatingoats,oats,2-day-old
2-day-oldseedlings
seedlingswere
weretreated
treatedwith
with various
various MeJA
MeJA
concentrations
concentrationsfor for33days.
days.The Theaccumulation
accumulationofofavenanthramides
avenanthramides inin
the seedlings
the seedlings gradu-
grad-
ally increased
ually increased along with
along the MeJA
with the MeJAconcentration, reaching
concentration, a peakaatpeak
reaching 75 μM
at MeJA
75 µM(578.61
MeJA
mg/kg
(578.61FW),
mg/kgwhich
FW),was whicha 2.3-fold increaseincrease
was a 2.3-fold relativerelative
to that to
in that
control oats and
in control oatsresulting in
and result-
ing in Avn A, B, and C contents of 179.8 mg/kg FW, 268.7 mg/kg FW,
Avn A, B, and C contents of 179.8 mg/kg FW, 268.7 mg/kg FW, and 130.0 mg/kg FW, re- and 130.0 mg/kg FW,
respectively
spectively (Figure
(Figure 4B).
4B). ToTodetermine
determinethe theoptimal
optimalconcentration
concentrationofofABA, ABA,2-day-old
2-day-old oats
oats
weretreated
were treated with
withvarious
variousABA ABAconcentrations
concentrations (range:
(range: 10–300
10–300µµM), andwhole
M), and wholeplants
plantswere
were
collectedon
collected onday
day33after
aftertreatment.
treatment.Figure
Figure4C 4Cshows
showsthat
thatABA
ABAtreatment
treatmentat atall
allconcentrations
concentrations
significantlyincreased
significantly increasedthe theproduction
productionof ofavenanthramides
avenanthramidesin inthe
the germinating
germinatingoats oats relative
relative
to the control. In particular, Avn C production increased >4.0-fold at all
to the control. In particular, Avn C production increased >4.0-fold at all concentrations, sug-concentrations,
suggesting
gesting that that
ABAABA played
played an important
an important role inrole
Avnin CAvn C accumulation.
accumulation. Moreover,
Moreover, com-
compared
pared with the control, the maximum level of produced avenanthramides
with the control, the maximum level of produced avenanthramides was observed from ger- was observed
from germinating
minating oats treated oatswith
treated with
50 μM ABA50 µM ABA (aincrease;
(a 3.4-fold 3.4-fold increase;
828.9 mg/kg 828.9 mg/kg FW).
FW).

Figure 4. Effect of MeJA or ABA concentration on avenanthramide production in germinating oats.

Figure 4. Effect
Two-day-old of MeJA oroats
germinating ABA concentration
were treated with ondifferent
avenanthramide production
concentrations in or
of MeJA germinating
ABA for 3 days.
oats. Two-day-old germinating oats were treated with different concentrations of
(A) Results of HPLC analysis with UV detection at 340 nm. From top to bottom, chromatogramsMeJA or ABA for 3of
days. (A) Results of HPLC analysis with UV detection at 340 nm. From top to bottom, chromato-
STDs, control oats, and oats treated with MeJA (75 µM) and/or ABA (50 µM). The chromatogram
grams of STDs, control oats, and oats treated with MeJA (75 µM) and/or ABA (50 µM). The chroma-
of the STD samples shows retention times of 15.511 min, 17.320 min, and 10.964 min for the Avn A,
togram of the STD samples shows retention times of 15.511 min, 17.320 min, and 10.964 min for the
Avn
Avn A,B, Avn
and Avn C STDs,
B, and Avn Crespectively. (B,C) Quantitative
STDs, respectively. measurement
(B,C) Quantitative of avenanthramide
measurement levels in
of avenanthramide
germinating oats treated with (B) MeJA or (C) ABA. Ethanol (0.1%) was used as the control.
levels in germinating oats treated with (B) MeJA or (C) ABA. Ethanol (0.1%) was used as the control. MeJA,
methylmethyl
MeJA, jasmonate; ABA, ABA,
jasmonate; abscisic acid; Avn,
abscisic acid; avenanthramide; STD, STD,
Avn, avenanthramide; standard. Different
standard. letters
Different above
letters
above theindicates
the bars bars indicates significant
significant differences
differences determined
determined by ANOVAby ANOVA
followedfollowed by a test
by a Tukey’s Tukey’s test
(p < 0.05).
(p < 0.05).
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2.4. Synergistic
2.4. Effect
Synergistic of of
Effect MeJA and
MeJA ABA
and Co-Treatment
ABA Co-TreatmentononAvenanthramide
AvenanthramideProduction
Productioninin
Germinating
GerminatingOats
Oats
The
Theresults
results demonstrated
demonstrated that MeJA
that MeJA oror
ABA
ABA cancaninduce
induce thethe
production
production of of
avenan-
avenan-
thramides in geminating oats. Therefore, further experiments were conducted
thramides in geminating oats. Therefore, further experiments were conducted to determine to deter-
mine whether co-treatment with MeJA and ABA might have a synergistic
whether co-treatment with MeJA and ABA might have a synergistic effect on avenan- effect on av-
enanthramide production (Figure 5). Two-day-old germinating oats
thramide production (Figure 5). Two-day-old germinating oats were treated for 3 days were treated for 3
days
withwith various
various concentrations
concentrations of MeJA
of MeJA andand
ABAABA based
based ononthethe concentrations
concentrations usedused
in in
the
the experimentsreported
experiments reportedininFigure
Figure3.3. Combined
Combined MeJA MeJA and andABAABAtreatment
treatmentincreased
increasedthe the
production
production ofofavenanthramides
avenanthramides atat
allall
concentrations
concentrations tested.
tested.Specifically,
Specifically,compared
compared with
with
the control,
the the
control, thehighest
highestcombined
combinedyields
yieldsofofAvn
AvnAA(515.6
(515.6mg/kg
mg/kgFW), FW),Avn
AvnBB(549.9
(549.9mg/kg
mg/kg
FW),
FW),and
andAvn
AvnCC(439.8
(439.8mg/kg
mg/kg FW)FW)were
were observed
observedin in
oats treated
oats treatedwith
with 7575
μMµM MeJA
MeJA and
and
2525
μMµMABA
ABA (Figure
(Figure5B). These
5B). These results
resultsconfirmed
confirmedthat
thatcombined
combined treatment
treatment with
withMeJA
MeJA and
and
ABA
ABA had
had a synergistic
a synergistic effect
effect onon increasing
increasing avenanthramide
avenanthramide synthesis
synthesis in in germinating
germinating oats.
oats.

Figure
Figure5. 5.
Enhanced
Enhanced production
production of avenanthramides
of avenanthramides by by
combined
combinedtreatment with
treatment MeJA
with andand
MeJA ABA.ABA.
Two-day-old germinating oats were treated with different concentrations of MeJA
Two-day-old germinating oats were treated with different concentrations of MeJA and ABA and ABA for 3 for
days. (A) Results
3 days. of HPLC
(A) Results analysis
of HPLC withwith
analysis UV detection at 340
UV detection atnm.
340 Chromatograms
nm. Chromatograms of STDs (top),(top),
of STDs
control oats (middle), and oats receiving combined treatment (75 µ M MeJA + 25 µ M ABA). The
control oats (middle), and oats receiving combined treatment (75 µM MeJA + 25 µM ABA). The
chromatogram of the STD samples shows retention times of 15.511 min, 17.320 min, and 10.964
chromatogram of the STD samples shows retention times of 15.511 min, 17.320 min, and 10.964 min
min for the Avn A, Avn B, and Avn C STDs, respectively. (B) Avenanthramide levels in germinat-
ingfor thereceiving
oats Avn A, Avn B, and Avn
combined C STDs,
treatment respectively.
with MeJA and (B) Avenanthramide
ABA. Ethanol (0.1%)levels in germinating
was used as the con- oats
receiving
trol. combined
MeJA, methyl treatment
jasmonate; withabscisic
ABA, MeJA and
acid;ABA.
Avn,Ethanol (0.1%) wasSTD,
avenanthramide; usedstandard.
as the control. MeJA,
Different
methyl
letters jasmonate;
above the barsABA, abscisic
indicates acid; Avn,
significant avenanthramide;
differences STD,
determined bystandard. Different by
ANOVA followed letters
a above
Tukey’s test
the bars (p < 0.05).
indicates significant differences determined by ANOVA followed by a Tukey’s test (p < 0.05).

2.5.
2.5. Expression
Expression of of Avenanthramide-Biosynthesis-Related
Avenanthramide-Biosynthesis-Related Genes
Genes inin MeJA-and
MeJA- andABA-Co-Treated
ABA-Co-Treated Oats
Oats
The expression of avenanthramide-biosynthesis-related genes was further compared
The expression of avenanthramide-biosynthesis-related genes was further compared
after single treatments with MeJA (75 µM) or ABA (25 µM) and combined treatments with
after single treatments with MeJA (75 μM) or ABA (25 μM) and combined treatments with
MeJA (75 µM) and ABA (75 µM). Oat samples treated with 0.1% EtOH were used as a
MeJA (75 μM)
negative and and
control, ABAqRT-PCR
(75 μM).wasOatperformed
samples treated with 0.1%
on 5-day-old EtOH were
germinating oatsused as a6).
(Figure
negative
qRT-PCR results showed synergistic upregulation of AsPAL, As4CL, and AsHHT56).ex-
control, and qRT-PCR was performed on 5-day-old germinating oats (Figure
qRT-PCR
pression results
following showed synergistic
co-treatment upregulation
of germinating oatsofwith
AsPAL,
MeJAAs4CL,
and ABA,andwith
AsHHT5
AsPAL
expression following co-treatment of germinating oats with MeJA and ABA,
expression upregulated 2.5- and 2.8-fold by MeJA and ABA treatment, respectively, and with AsPAL
expression
4.0-fold byupregulated
co-treatment 2.5-relative
and 2.8-fold
to the by MeJABy
control. and ABA treatment,
contrast, respectively,
As4CL expression and
increased
4.0-fold by co-treatment relative to the control. By contrast, As4CL expression
only slightly by 1.5-fold in response to co-treatment. Furthermore, among the six AsHHTs increased
only slightly
genes by 1.5-fold
analyzed in thisinstudy,
response to co-treatment.
AsHHT5 Furthermore,
exhibited the among thewith
highest expression, six AsHHTs
a 5.1-fold
genes analyzed in this study, AsHHT5 exhibited the highest expression,
increase following co-treatment and 1.6- and 3.1-fold increases following separate with a 5.1-fold
MeJA
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 of 15

Int. J. Mol. Sci. 2021, 22, 4779 8 of 15

increase following co-treatment and 1.6- and 3.1-fold increases following separate MeJA
and ABA treatments, respectively, relative to the control. However, the expression of
AsHHT1, AsHHT2, AsHHT3, AsHHT4, and AsHHT6 was reduced by separate MeJA or
and ABA
ABA treatments,
treatment, respectively,
and even relative
co-treatment toto
failed the control.their
increase However, the relative
expression expression of
to the
AsHHT1, AsHHT2, AsHHT3, AsHHT4, and AsHHT6 was reduced by separate
control. These results suggested that among the six AsHHTs genes, AsHHT5 played a MeJA or ABA
treatment,
more androle
crucial evenin
co-treatment failed tobiosynthesis
avenanthramide increase theirinexpression
response relative
to MeJA to the
andcontrol.
ABA
These results suggested that among the six AsHHTs genes, AsHHT5 played
treatment. Additionally, it was found that AsHHT1 expression was relatively high a more crucial
as
role in avenanthramide biosynthesis in response to MeJA and ABA treatment.
compared with that of the other AsHHT genes in the untreated germinating oats. In Additionally,
it was found that AsHHT1 expression was relatively high as compared with that of the
particular, AsHHT3 and AsHHT4 expression was undetectable in germinating oats,
other AsHHT genes in the untreated germinating oats. In particular, AsHHT3 and AsHHT4
regardless of treatment status (with MeJA and ABA or untreated) (Figure S1). These
expression was undetectable in germinating oats, regardless of treatment status (with MeJA
findings suggested that the six AsHHT genes were effectively upregulated in response to
and ABA or untreated) (Figure S1). These findings suggested that the six AsHHT genes
high concentrations of MeJA (100 μM) and ABA (300 μM), whereas five AsHHT genes
were effectively upregulated in response to high concentrations of MeJA (100 µM) and
(except AsHHT5) failed to respond to relatively low concentrations of MeJA (75 μM) and
ABA (300 µM), whereas five AsHHT genes (except AsHHT5) failed to respond to relatively
ABA (25 μM).
low concentrations of MeJA (75 µM) and ABA (25 µM).

Figure 6. The relative expression

expression of
of avenanthramide-biosynthetic
avenanthramide-biosyntheticgenesgenesfollowing
followingseparate
separateand
andcom-
combined treatment
bined treatment withwith
MeJAMeJA and ABA
and ABA of germinating
of germinating oats.oats. Transcript
Transcript levelslevels of PAL,
of PAL, 4CL,
4CL, CCoAOMT,
CCoAOMT,
HHT1, HHT2, HHT1,
HHT3,HHT2,
HHT4,HHT3,
HHT5,HHT4, HHT5,
and HHT6 and
were HHT6 were
analyzed analyzedinby
by qRT-PCR qRT-PCR in
germinating germi-
oats treated
with 75 µM MeJA and 25 µM ABA for 3 days. Relative expression levels were normalized to were
nating oats treated with 75 μM MeJA and 25 μM ABA for 3 days. Relative expression levels that of
normalized to that of AsActin (KP257585) and are presented as fold induction relative to the con-
AsActin (KP257585) and are presented as fold induction relative to the control. Data represent the
trol. Data represent the mean of three independent replicates ± SD. Student’s t-test; * p < 0.05. qRT-
mean of three independent replicates ± SD. Student’s t-test; * p < 0.05. qRT-PCR, quantitative reverse
PCR, quantitative reverse transcription PCR; SD, standard deviation; MJ, methyl jasmonate.
transcription PCR; SD, standard deviation; MJ, methyl jasmonate.
2.6.
2.6. Analysis
Analysis of
of Avenanthramide Contents and
Avenanthramide Contents and Biosynthetic
Biosynthetic Gene
Gene Expression
ExpressionininDifferent
DifferentOrgans
Organs of Germinating
of Germinating Oats Oats
The total contents of all three avenanthramides in different organs 3 days following
co-treatment of 2-day-old germinating oats with MeJA and ABA as elicitors were further
analyzed. Different
Different organ
organ samples
samples (leaf,
(leaf, grain, and root) were collected from
from 5-day-old
5-day-old
oat seedlings after
after co-treatment
co-treatmentandandwere
weredetermined
determinedusing
usingHPLC.
HPLC.As Asshown
shownininTable
Table
1, 1,
a
ahigher
highercontent
contentofofavenanthramides
avenanthramideswas wasdetected
detectediningrains
grains(420.7
(420.7mg/kg
mg/kg FW)
FW) relative to
root (2.3 mg/kg
mg/kg FW)
FW) andand leaf (13.2 mg/kg
mg/kg FW)FW) organs.
organs. By
By contrast,
contrast, total avenanthramide
content was significantly increased by co-treatment with MeJA and ABA, with the avenan-
thramide contents in co-treated roots (15.6 mg/kg FW) and leaves (40.1 mg/kg FW) ~6.7-
Int. J. Mol. Sci. 2021, 22, 4779 9 of 15

and ~3.0-fold higher than those in untreated roots and leaves, respectively. The highest
content of all three avenanthramides (968.9 mg/kg FW) was observed in the co-treated
grains, which was ~2.3-fold higher relative to that in untreated grains (420.7 mg/kg FW).
Among the three avenanthramides examined in this study, Avn B was the most abundant
(~46%) in co-treated grains (Avn A, ~32%; and Avn C, ~21%) (Table 1). Overall, these results
showed that avenanthramide content was increased throughout the plant by combined
treatment with MeJA and ABA, with the highest avenanthramide production observed in
the grain.
To determine the relationship between avenanthramide production and its biosyn-
thetic gene expression, the relative expression levels of avenanthramide-biosynthesis-
related genes in untreated and co-treated oat tissues were compared (Figure S2). Among
the three tissues, the highest expression of AsPAL, As4CL, AsHHT1, AsHHT2, AsHHT5,
and AsHHT6 was observed in the grains, whereas AsCCoAOMT expression was higher in
root tissues relative to the control. This was positively correlated with the high avenan-
thramide production observed in the grains (Table 1). Additionally, the expression of
AsPAL, As4CL, AsCCoAOMT, and AsHHTs following co-treatment with MeJA (75 µM)
and ABA (25 µM) relative to the untreated control was examined (Figure 7). The results
showed that AsPAL was upregulated ~5.4-, ~2.0-, and ~10.0-fold in leaf, grain, and root
tissues, respectively. Moreover, As4CL expression was upregulated ~1.6-fold in grains
but reduced ~0.6-fold in roots following co-treatment, and AsCCoAOMT expression was
decreased ~0.6-fold in co-treated roots relative to the control. Furthermore, AsHHT6 ex-
pression was slightly increased by ~1.5- and ~1.2-fold in leaves and roots, respectively, but
reduced ~0.4-fold in grains following co-treatment and relative to the control. AsHHT5
expression was upregulated ~2.3-fold in grains but downregulated ~0.7-fold in roots rela-
tive to the control. However, AsHHT2, AsHHT3, and AsHHT4 expression was reduced
following co-treatment in all the tissues examined, with AsHHT3 and AsHHT4 expression
decreased by ~0.4- and ~0.3-fold, respectively, relative to the control. By contrast, AsHHT1
expression was slightly upregulated by ~1.3-fold in leaves but decreased ~0.4-fold in roots
relative to the control. Among the six AsHHTs, the greatest increase in expression in
response to co-treatment was observed in AsHHT5 in grains; therefore, this suggested that
AsHHT5 might play a more important role in avenanthramide biosynthesis in germinating
oats in response to MeJA and ABA co-treatment. Furthermore, this result was consistent
with the highest contents of all three avenanthramides in co-treated grains (Table 1).

Table 1. Analysis of avenanthramide contents in different organs of germinating oats.

Total
Avn A Avn B Avn C
Organs Avenanthramides
(mg/kg FW) (mg/kg FW) (mg/kg FW)
(mg/kg FW)
Leaves 2.9 ± 1.7 a 6.2 ± 0.4 a 4.1 ± 2.1 a 13.2 ± 3.9 a
Untreated
Grains 112.4 ± 22.7 b 235.9 ± 41.5 b 72.5 ± 23.8 a 420.7 ± 85.1 b
control
Roots 0±0a 2.3 ± 2.3 a 0±0a 2.3 ± 2.3 a
Leaves 9.2 ± 2.9 ab 21.8 ± 8.5 a 9.1 ± 0.2 a 40.1 ± 11.5 a
MeJA + ABA * Grains 312.7 ± 51.1 c 449.1 ± 78.6 c 207.1 ± 31.7 b 968.9 ± 160.7 c
Roots 3.9 ± 1.9 ab 10.1 ± 5.1 a 1.6 ± 1.6 a 15.6 ± 8.1 a
* Combined treatment with 75 µM MeJA and 25 µM ABA. Statistical differences among experimental conditions are labeled with different
letters (p < 0.05).
Mol. Sci. 2021,
Int. 22, x FOR
J. Mol. Sci.PEER
2021,REVIEW
22, 4779 10 of 15 10 of 15

Figure 7. TheFigure 7. The

relative relative levels
expression expression levels of avenanthramide-biosynthetic
of avenanthramide-biosynthetic genestypes
genes in three in three types
of oat of oat(A) leaves,
organs:
organs: (A) leaves, (B) grains, and (C) roots. Combined treatment (MeJA + ABA) comprised 75 μM
(B) grains, and (C) roots. Combined treatment (MeJA + ABA) comprised 75 µM MeJA and 25 µM ABA. Two-day-old
MeJA and 25 μM ABA. Two-day-old germinating oats were treated for 3 days and different or-
germinating oats were treated for 3 days and different organs were collected as leaves, grains, and roots. Transcript levels of
gans were collected as leaves, grains, and roots. Transcript levels of PAL, 4CL, CCoAOMT, HHT1,
PAL, 4CL, CCoAOMT,
HHT2, HHT3,HHT1, HHT2,
HHT4, HHT3,and
HHT5, HHT4,
HHT6 HHT5,
wereand HHT6 by
analyzed were analyzedRelative
qRT-PCR. by qRT-PCR. Relative
expression expression levels
levels
were normalized
were normalized against AsActin (KP257585) and are presented as fold induction relative to therepresent the
against AsActin (KP257585) and are presented as fold induction relative to the control. Data
mean of threecontrol.
independent
Data represent ± SD.
replicatesthe mean of threet-test;
Student’s * p < 0.05.replicates
independent qRT-PCR,± quantitative
SD. Student’s reverse
t-test; transcription
* p < 0.05. PCR; SD,
standard deviation;
qRT-PCR,MJ,quantitative
methyl jasmonate.
reverse transcription PCR; SD, standard deviation; MJ, methyl jasmonate.

3. Discussion3. Discussion
Oats have gainedOatsconsiderable
have gainedinterest
considerable interest
in the past decadein the
duepast decade
to their due tosig-
nutritional their nutritional
nificance and significance and health-beneficial
health-beneficial effects, mainly aseffects, mainly
foodstuffs butasalso
foodstuffs but also as pharmaceuti-
as pharmaceuticals
cals [5,13,17,32].
[5,13,17,32]. Several Several
studies report thatstudies report that have
avenanthramides avenanthramides have antioxidant
antioxidant properties and properties
and potential
potential therapeutic therapeutic
benefits, includingbenefits, including anti-inflammatory,
anti-inflammatory, antiproliferative, andantiproliferative,
anti- and
antigenotoxic effects [11,13,15,17,19,33]. These health benefits
genotoxic effects [11,13,15,17,19,33]. These health benefits make avenanthramides attrac- make avenanthramides at-
tractive for their potential use in cosmetic, nutraceutical, and therapeutic
tive for their potential use in cosmetic, nutraceutical, and therapeutic preparations. There- preparations.
Therefore,
fore, it is important it is important
to enhance to enhance
their levels their
in oats in levels
order in oats inconsumers
to provide order to provide
with rawconsumers with
raw materials
materials of enhanced of enhanced
nutritional value.nutritional value.
In this study, In this study, high-value-added
high-value-added oats capable of oats capable
Int. J. Mol. Sci. 2021, 22, 4779 11 of 15

of producing high levels of avenanthramides following the application of elicitor-mediated

metabolite-farming technology were developed. The application of exogenous elicitors
often causes an increase in the concentration of bioactive compounds in plants [3,27,28].
MeJA, ABA, SA, and ethylene are currently used as powerful elicitors to induce secondary
metabolism in plants and plant cell cultures. For example, preharvest treatment of ethy-
lene on soybean plants induces significant accumulation of dietary isoflavones (up to
13,854 mg/g DW) in the leaves [28]. Therefore, elicitor-mediated metabolite farming
can be a valuable agricultural technology enabling increases in the amounts of bioactive
metabolites generated in plants or food crops.
In this study, the highest content of total avenanthramides was detected on day 3 in
germinating oats, with this content most abundant in grains and lowest in roots (Figure 2B
and Table 1). This was why seedlings were used as the experimental stage for evaluating
elicitor-mediated increases in avenanthramide content. Previous studies have reported
that the majority of avenanthramides are present in the oat groat, with the highest content
in the bran [34]. The biosynthesis of avenanthramides is known to increase during the ger-
mination process of oat grains [19,23,34]. Moreover, these compounds are shown to induce
in response to pathogen infection and elicitor treatment [5,7,9]. Wise et al. [9] previously
reported that avenanthramide levels in leaves and roots were enhanced by treatment of
oat plants with benzothiadiazole (BTH) via root drench, with BTH application eliciting
maximal levels (562.44 mg/kg FW) of total avenanthramide production in oat leaves.
Additionally, it was found that co-treatment with MeJA and ABA of germinating oats
synergistically induced the highest levels of avenanthramide production (1505.3 mg/kg
FW), which was substantially higher than those reported previously [35–37]. For example,
Chu et al. [35] reported that the total content of avenanthramides generated in whole
oat extracts ranged from 8.41 mg/kg to 150.81 mg/kg in varieties of oats from Canada
(1.05–43.77 mg/kg for Avn A; 3.66–58.63 mg/kg for Avn B; and 3.7–48.41 mg/kg for Avn C).
Tong et al. [36] reported that the total content of produced avenanthramides ranged from
4.4 mg/kg DW to 718.5 mg/kg DW in varieties of oats from China. Moreover, Li et al. [37]
reported that the total concentration of avenanthramides produced in whole oat grains
ranged between 22.1 mg/kg DW and 471.2 mg/kg DW, with the Avn B concentration
(7.3–222.8 mg/kg DW) in oat grains higher than that of the other two avenanthramides
(6.1–112.3 mg/kg DW for Avn A; and 6.2–136.2 mg/kg DW for Avn C). These results are in
agreement with the ones in the present study, where Avn B comprised the largest fraction
in both untreated and co-treated germinating oats (Table 1). By contrast, Wise et al. [9]
reported that Avn A was the most highly produced avenanthramide of the four analyzed,
with a maximum concentration of ~379 mg/kg FW in BTH-treated leaves. However, reports
showed that the concentration and composition of avenanthramides are greatly influenced
by genotype, growing environment, and growth conditions [24,34–37]. Furthermore,
although BTH strongly upregulated avenanthramide production in oat plants [9], no
significant effects of BTH application on oats in samples analyzed were identified in the
present study (data not shown). These different responses to BTH might be the result of
genotypic differences or different cultivars. Specifically, the naked oat cultivar was used in
the present study, whereas the husked oat cultivar was used by Wise et al. [9]. Based on
these findings, it can be speculated that BTH can induce avenanthramide biosynthesis in
husked cultivars but not in naked ones. Therefore, future research is needed to describe the
metabolic changes in different oat cultivars and reveal their underlying molecular causes.
Moreover, additional investigations are necessary to determine whether avenanthramide
accumulation is elevated in leaf tissues at different growth stages in response to BTH.
Although BTH is an effective elicitor for increasing avenanthramide production in leaves of
husked oat cultivars, because it is classified as a pesticide, its use is restricted, whereas the
use eco-friendly elicitors for healthy food products is promoted. As a result, it is important
to identify novel harmless elicitors for increasing the concentration of specific metabolites
in food crops.
Int. J. Mol. Sci. 2021, 22, 4779 12 of 15

Several studies show that Avn C exerts greater antioxidant activity than Avn A and
B [16–18,38,39]. Dhakal et al. [38] reported that Avn C from germinated oats exhibits
anti-allergic and anti-inflammatory effects in mast cells, and Umugire et al. [39] showed
that Avn C exerts a strong protective effect on hearing loss caused by noise trauma. These
results suggest Avn C as a promising candidate as a therapeutic for treating allergic diseases
and hearing damage, given that Avn C can be provided through dietary products derived
from oats. To this end, the CRISPR/Cas9 gene-editing system can potentially be applied to
create high-value-added oats with high Avn C content. Li et al. [21] recently demonstrated
the molecular mechanisms associated with biosynthesis of three major avenanthramides
in oats, suggesting that oat HHTs are only involved in the biosynthesis of Avn A and C
but not Avn B, which is synthesized by oat CCoAOMT enzyme from O-methylation of
Avn C. In the present study, it was found that the expression of AsHHT genes was induced
by MeJA and ABA treatment, respectively, especially AsHHT4 for MeJA and AsHHT5 for
ABA, thereby enhancing the production of all three avenanthramides in germinating oats.
Furthermore, we found that the basal expression level of AsCCoAOMT was high (~0.1
to 0.2-fold expression relative to AsActin) in the leaves, grains, and roots of germinating
oats regardless of elicitation (Figure S2), indicating constitutive gene expression. These
findings suggest that oat CCoAOMT might be a good target for gene editing to increase
Avn C content; however, the complete biosynthetic pathway of avenanthramides in oats
remains to be elucidated.
In conclusion, this study provides a basis for elicitor-mediated metabolite farming to
produce high-value-added oats capable of producing high levels of avenanthramides. The
data suggested that germinating oats co-treated with MeJA and ABA were the most effec-
tive producers of all three avenanthramides (Avn A, B, and C) and demonstrated a strong
correlation between enhancement of avenanthramide production and biosynthesis-gene
activation (AsPAL, As4CL, and AsHHT5) in germinating oats. Therefore, this metabolite-
farming method could also be applied to develop other high-value-added plants capable
of generating high levels of bioactive compounds with possible applications in functional
foods or pharmaceutical supplements.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions
The naked oat cultivar Choyang was obtained from the National Institute of Crop
Science, Development Administration in Korea. For the experiments, oat seeds were soaked
in water for 12 h and then sterilized at 50 ◦ C for 10 min in a water bath before being washed
three times with autoclaved distilled water (DW) [40]. Sterilized oat seeds were sown in
plant culture dishes (100 × 40 mm; SPL Life Sciences, Seoul, Korea) containing 7 mL of
water and two sheets of filter paper and cold-stratified at 4 ◦ C in the dark for 2 days. The
seeds were incubated at 25 ◦ C for germination with a 16 h/8 h light/dark cycle.

4.2. Treatment with Various Elicitors and Sample Collection

Two-day-old germinating oats (20 seedlings) were treated for 72 h with various
elicitors, as previously described [29]. In brief, MeJA and ABA were dissolved in 96% (v/v)
ethanol (EtOH), whereas MV, SA, ET, and Chi were dissolved in distilled water (DW). The
elicitors of 10 µM MV, 100 µM MeJA, 300 µM ABA, 100 µM SA, 300 µM ET, and 200 µg/mL
Chi were used. EtOH (0.1%) and DW were used as negative controls. Elicitor treatments
were performed in plant culture dishes containing 7 mL of elicitor solution with two sheets
of filter paper. The treated samples were harvested after 3 days of treatment, followed by
immediate freezing in liquid nitrogen. The samples were then pulverized using a mortar
and pestle for quantification of avenanthramides by HPLC and qRT-PCR analyses.

4.3. Quantification of Avenanthramides by HPLC

Avenanthramides were extracted by sonication (WUC-D10H; DAIHAN Scientific,
Wonju, Korea) for 20 min from 0.1 g of sample (FW) in 1 mL of 80% (v/v) methanol at
Int. J. Mol. Sci. 2021, 22, 4779 13 of 15

50 ◦ C. Following centrifugation at 11,000 rpm for 5 min, the supernatants were air-dried
by nitrogen evaporation (N-EVAP 111; Organomation Associates, Inc., Berlin, MA, USA).
The residue was dissolved in 200 µL of 80% methanol and filtered through a 0.2 µm PTFE
membrane [34]. HPLC analysis was performed at 30 ◦ C using an Agilent 1260 system
equipped with a quaternary pump, a ZORBAX SB-C18 column (5 µM, 4.6 × 150 mm),
and photodiode array detector (Agilent Technologies, Santa Clara, CA, USA). The mobile
phase comprised buffer A (H2O with 5% acetonitrile and 0.1% formic acid) and buffer B
(acetonitrile with 0.1% formic acid). A gradient in B of 13% to 30% over 30 min at a flow
rate of 1 mL/min was employed [22] with an injection volume of 5 µL and a detection
wavelength set to 340 nm. Standard avenanthramides purchased from Sigma-Aldrich
(St. Louis, MI, USA) (#30366, Avn A; #93105, Avn B; and #36465, Avn C) were used for
identification and quantification.

4.4. RNA Extraction and qRT-PCR Analysis

Harvested oat samples of whole seedlings or each organ (leaf, grain, and root) were
finely ground using a mortar and pestle with liquid nitrogen. Total RNA was extracted
via a modified version of the method described by Porebski et al. [41] using cetyltrimethy-
lammonium bromide (Intron Biotechnology, Seongnam, Korea) and an RNA extraction
kit (QIAGEN, Hilden, Germany). RNA concentration and purity were estimated by spec-
trophotometry (Nanodrop NS-11; DeNovix, Wilmington, DE, USA). qRT-PCR analysis was
performed using an iQ SYBR Green supermix reagent (Bio-Rad Laboratories, Hercules,
CA, USA) on a CFX96 real-time PCR system (Bio-Rad Laboratories). Specific primers
(Table S1) were designed, as described by Yang et al. [8]. The reactions were subjected
to an initial denaturation step at 95 ◦ C for 15 min, followed by 40 cycles of denaturation
at 95 ◦ C for 20 s and annealing at 60 ◦ C for 40 s, with melting curve analysis performed
within the same tube or plate. Transcript levels were calculated by normalization against
AsActin (KP257585) as a reference control. Relative changes in gene-expression levels were
determined by the 2−∆∆Ct method [42].

4.5. Statistical Analysis

Data were analyzed using SPSS (v.22.0; IBM Corp., Armonk, NY, USA). One-way
analysis of variance was performed and followed by Tukey’s multiple range post hoc test
to compare data among experimental groups. Comparisons of the mean values between
two groups were performed using Student’s t-tests. All values are expressed as the mean
± standard deviation of triplicate experiments, and a p value < 0.05 was considered
statistically significant.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/ijms22094779/s1, Table S1: Primer pairs used for qRT-PCR in this study, Figure S1: The relative
expression of avenanthramide-biosynthetic genes following co-treatment with MeJA and ABA of
germinating oats, Figure S2: The relative expression levels of avenanthramide-biosynthetic genes in
three types of oat tissues: (A) leaves, (B) grains, and (C) roots.
Author Contributions: C.Y.K., J.C.J. and K.Y.Y. conceived and supervised this study; C.Y.K., J.C.J.,
S.K. and Y.J.J. designed the experiments; T.H.K., S.K., Y.J.J. and S.H.P. performed the treatment of
elicitors; T.H.K., J.L., S.C.P. and S.K. performed the qRT-PCR experiments; T.H.K., S.K. and Y.J.J.
performed the HPLC experiments; C.Y.K., J.C.J., T.H.K. and S.K. wrote the manuscript with assistance
from the other authors. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by grants from the KRIBB Initiative Program and the Next-
Generation BioGreen 21 Program (SSAC, grant no.: PJ01318604), Rural Development Administration,
Republic of Korea.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Int. J. Mol. Sci. 2021, 22, 4779 14 of 15

Data Availability Statement: The data presented in this study are available on request from the
corresponding author. Supplementary materials can be found at https://zenodo.org/record/4682677
(accessed on 13 April 2021).
Acknowledgments: We thank Byong Won Lee, National Institute of Crop Science (NICS), Rural
Development Administration (RDA) for kindly providing Choyang seeds for the experiments.
Conflicts of Interest: The authors declare no conflict of interest.

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