plants

Article
Morphological, Biochemical, and Proteomic Analyses to
Understand the Promotive Effects of Plant-Derived Smoke
Solution on Wheat Growth under Flooding Stress
Setsuko Komatsu 1, * , Hisateru Yamaguchi 2 , Keisuke Hitachi 3 , Kunihiro Tsuchida 3 , Shafiq Ur Rehman 4
and Toshihisa Ohno 1

1 Faculty of Life and Environmental Sciences, Fukui University of Technology, Fukui 910-8505, Japan;
ohno@fukui-ut.ac.jp
2 Department of Medical Technology, Yokkaichi Nursing and Medical Care University,
Yokkaichi 512-8045, Japan; h-yamaguchi@y-nm.ac.jp
3 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-1192, Japan;
hkeisuke@fujita-hu.ac.jp (K.H.); tsuchida@fujita-hu.ac.jp (K.T.)
4 Department of Biology, University of Haripur, Haripur 22620, Pakistan; drshafiq@yahoo.com
* Correspondence: skomatsu@fukui-ut.ac.jp; Tel.: +81-276-29-2466

Abstract: Wheat is an important staple food crop for one-third of the global population; however, its
growth is reduced by flooding. On the other hand, a plant-derived smoke solution enhances plant
growth; however, its mechanism is not fully understood. To reveal the effects of the plant-derived
smoke solution on wheat under flooding, morphological, biochemical, and proteomic analyses were
conducted. The plant-derived smoke solution improved wheat-leaf growth, even under flooding.
According to the functional categorization of proteomic results, oppositely changed proteins were
correlated with photosynthesis, glycolysis, biotic stress, and amino-acid metabolism with or without
Citation: Komatsu, S.;
the plant-derived smoke solution under flooding. Immunoblot analysis confirmed that RuBisCO
Yamaguchi, H.; Hitachi, K.;
Tsuchida, K.; Rehman, S.U.; Ohno, T.
activase and RuBisCO large/small subunits, which decreased under flooding, were recovered by the
Morphological, Biochemical, and application of the plant-derived smoke solution. Furthermore, the contents of chlorophylls a and b
Proteomic Analyses to Understand significantly decreased by flooding stress; however, they were recovered by the application of the
the Promotive Effects of plant-derived smoke solution. In glycolysis, fructose-bisphosphate aldolase and glyceraldehyde-3-
Plant-Derived Smoke Solution on phosphate dehydrogenase decreased with the application of the plant-derived smoke solution under
Wheat Growth under Flooding Stress. flooding as compared with flooding alone. Additionally, glutamine, glutamic acid, aspartic acid,
Plants 2022, 11, 1508. https:// and serine decreased under flooding; however, they were recovered by the plant-derived smoke
doi.org/10.3390/plants11111508 solution. These results suggest that the application of the plant-derived smoke solution improves
Academic Editor: Pavel Kerchev the recovery of wheat growth through the regulation of photosynthesis and glycolysis even under
flooding conditions. Furthermore, the plant-derived smoke solution might promote wheat tolerance
Received: 10 April 2022
against flooding stress through the regulation of amino-acid metabolism.
Accepted: 3 June 2022
Published: 4 June 2022
Keywords: proteomics; wheat; plant-derived smoke solution; flooding stress
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Wheat is the most important staple crop, and its availability can impact the livelihoods
of almost every family globally [1]. Climate change is widely accepted and leads to
Copyright: © 2022 by the authors.
many extreme climatic events related to temperature, precipitation, and other climatic
Licensee MDPI, Basel, Switzerland. conditions [2]. Climate change is a significant challenge to the agricultural production of
This article is an open access article wheat both regionally and globally [3]. Due to high rainfall, irrigation practices, and poor
distributed under the terms and soil drainage, waterlogging annually affects large areas of farmlands worldwide, and these
conditions of the Creative Commons effects result in anoxic soils and severe hypoxia or anoxia within crop roots [4]. Hypoxia
Attribution (CC BY) license (https:// caused by waterlogging inhibited the growth of crop roots/stems and the yield of seeds [5].
creativecommons.org/licenses/by/ Because waterlogging tolerance is different among wheat varieties, its tolerance mechanism
4.0/). during wheat growth has not been elucidated.

Plants 2022, 11, 1508. https://doi.org/10.3390/plants11111508 https://www.mdpi.com/journal/plants

Plants 2022, 11, 1508 2 of 18

The plant-derived smoke solution is a material for promoting plant growth/development

and affects plant species from various habitats [6]. Plant-derived smoke positively affected
the post-germination growth of rice [7–9], maize [10], chickpea [11], soybean [12–15], and
wheat [16]. Studies on the post-germination of crops treated with the plant-derived smoke
solution elucidated that its treatment affected not only the seed-germination stage but
also the plant growth and development stages [17]. Butanolides, including karrikins and
cyanohydrin, are the active compounds in the plant-derived smoke solution [6]. The
functional mechanisms of plant-derived smoke in the seed-germination stage were clarified
with the discovery of karrikin [18]. A study on the molecular aspects of seed germination
reported that abscisic acid, seed maturation, and dormancy-related transcripts were up-
regulated by trimethyl butenolide and suppressed by karrikin 1, indicating that increased
seed germination by karrikin 1 might be due to suppression of abscisic acid [6,18]. However,
the role of karrikin is not elucidated for plant-growth stages.
It was reported that the plant-derived smoke solution enhanced soybean growth
under flooding [13] and after flooding [12]. Zhong et al. [13] reported that proteins related
to the ubiquitin-proteasome pathway were altered and led to the sacrifice-for-survival-
mechanism-driven degradation of the root tip in soybean by the plant-derived smoke
solution, which enabled the accumulation of metabolites and guaranteed lateral-root
development during soybean recovery from flooding. Li et al. [12] reported that the plant-
derived smoke solution enhanced soybean growth during recovery from flooding through
the balance of sucrose/starch metabolism and glycolysis and/or the accumulation of cell-
wall-related protein. On the other hand, in the case of wheat, the plant-derived smoke
treatment improved the shoot length under optimum conditions [16]; however, the mecha-
nism, which promotes plant growth, is not fully understood. In this study, to reveal the
dynamic effects of the plant-derived smoke solution on wheat under flooding, a morpho-
logical analysis was performed. Based on its result, proteomic analysis using nano-liquid
chromatography (LC) and mass spectrometry (MS)/MS was conducted. Furthermore, the
proteomic results were confirmed using immunoblot analysis, chlorophyll-contents assay,
and amino-acid analysis.

2. Results
2.1. Morphological Changes of Wheat Treated with Plant-Derived Smoke Solution under
Flooding Stress
To investigate the effect of the plant-derived smoke solution on wheat under flooding
stress, morphological analysis was performed. Wheat seeds were treated with 2000 ppm
of the plant-derived smoke solution, and the 3-day-old plant was flooded for 3 days
(Figure 1). As morphological parameters, leaf length, leaf-fresh weight, main-root length,
and total-root fresh weight were measured (Figure 2). All parameters decreased under
flooding; however, leaf length and leaf-fresh weight increased with the application of
the plant-derived smoke solution, even if it was under flooding (Figure 2). Based on the
morphological results, wheat leaves were used for proteomic analysis.

2.2. Protein Identification and Functional Categorization in Wheat Treated with Plant-Derived
Smoke Solution under Flooding Stress
To investigate the cellular mechanism in wheat growth by the application of the
plant-derived smoke solution under flooding stress, a gel-free/label-free proteomics was
conducted (Table S1). Three kinds of treatments, which were control, flood, and flood
+ smoke, were performed. Proteins extracted from wheat leaves after treatment were
enriched, reduced, alkylated, and digested. After analysis by LC combined MS/MS, the
relative abundance of proteins from without (Table S2) or with (Table S3) the plant-derived
smoke solution under flooding stress was compared to that from the control.
Totally, 5774 proteins were identified by LC-MS/MS analysis (Figure 3). The proteomic
results of all 9 samples from different 3 groups were compared by principal component
analysis (PCA), which showed the different accumulation patterns of proteins from three
different kinds of treatment (Figure 3). This result indicated that flooding stress largely
 Plants 2022, 11, 1508 3 of 18

Plants 2022, 11, x FOR PEER REVIEW 3 of 18

Plants 2022, 11, x FOR PEER REVIEW affected the wheat proteins; however, this effect was recovered at the protein
3 oflevel
18 by the
application of the plant-derived smoke solution, even if it was under flooding (Figure 3).

Figure 1. The experimental

Figure design for the
1. The experimental investigation
design of the effect of
for the investigation of the
the plant-derived smoke so-
effect of the plant-derived smoke
Figureon
lution 1. wheat
The experimental design
under flooding forTo
stress. theinvestigate
investigation of the effect
the potential of the
effects plant-derived
of the smoke
plant-derived so-
smoke
lution ononsolution
wheat on
under wheat under
flooding flooding stress. To investigate the potential effects of the plant-derived
solution wheat, seeds were stress.
sown and To investigate
treated with theorpotential
withouteffects
2000 of the of
ppm plant-derived smoke
the plant-derived
solution smoke
on solution
wheat, seeds on wheat,
were sown seeds
and were sown
treated with and
or treated 2000
without with ppm
or without
of the 2000 ppm of the plant-
plant-derived
smoke solution. After 3 days of sowing, wheat was flooded for 3 days. Wheat seedlings were ana-
smokewith
lyzed derived
solution. smoke
After
morphological solution.
3 days Afterwheat
of sowing,
and proteomic 3 days of sowing,
was
methods, flooded wheat was flooded
for 3 days.
and confirmation. Wheat for 3 days.
seedlings
For confirmation Wheat
were ana- seedlings
experi-
lyzed with morphological
were analyzed and
with proteomic
morphological methods,
and and
proteomic confirmation.
methods, For
and
ments, immunoblot and amino-acid analyses were used. All experiments were performed with confirmation
confirmation. experi-
For confirmation
ments, immunoblot
experiments,and amino-acid
immunoblot and
three independent biological replicates. analyses
amino-acidwere used.
analyses All experiments
were used. All were performed
experiments were with
performed with
three independent biological replicates.
three independent biological replicates.

Figure 2. The morphological effects of the plant-derived smoke solution on wheat under flooding
FigureWheat
stress. 2. The morphological
Figure 2. The
seeds sown effects
weremorphological of the
and treated plant-derived
effects
withof the smoke2000solution
plant-derived
or without of on
smoke
ppm wheat
solution
the under
on wheat
plant-derived flooding
under flooding
smoke
stress. Wheat seeds
solution. Three-day-oldwere sown
wheatswere
stress. Wheat seeds and treated
weresown
treated with
andwith or without
or without
treated 2000 ppm
with or flooding of the
for three
without 2000 plant-derived
ppmdays.
of theAs smoke smoke
morpho-
plant-derived
solution.
logical Three-day-old
parameters, wheatsleaf-fresh
were treated with or without flooding for three fresh
days. weight
As morpho-
solution. leaf length,
Three-day-old wheats weight, main-root
were treated withlength, and total-root
or without flooding for three were
days. As morpho-
logical parameters,
analyzed 6logical leaf
days after length,
sowing. leaf-fresh
The bar in weight,
the left main-root
panel length,
indicates 1 and
cm total-root
in the fresh
picture. weight
The datawere
are
parameters, leaf length, leaf-fresh weight, main-root length, and total-root fresh weight were
analyzed 6asdays
presented meanafter
± SD sowing. The independent
from three bar in the leftbiological
panel indicates 1 cmAsterisks
replicates. in the picture.
indicateThe data are
significant
presented analyzed
as mean 6 SD
± days after
from sowing.
three The bar biological
independent in the left replicates.
panel indicates 1 cmindicate
Asterisks in the picture. The data are
significant
changes between wheats treated with the plant-derived smoke solution under flooding and with
changes presented
between as mean ±
wheatstotreated SD from
with thethree independent
plant-derived biological replicates. Asterisks indicate significant
only flooding according the Student’s t-test (**: p < 0.01).smoke solution under flooding and with
changes between wheats treated with the
only flooding according to the Student’s t-test (**: p < 0.01). plant-derived smoke solution under flooding and with
only flooding according to the Student’s t-test (**: p < 0.01).
2.2. Protein Identification and Functional Categorization in Wheat Treated with Plant-Derived
2.2. Protein
Smoke Identification
Solution and Functional
under Flooding Stress Categorization in Wheat Treated with Plant-Derived
Smoke Solution under Flooding Stress
To investigate the cellular mechanism in wheat growth by the application of the
To investigate
plant-derived smokethe cellular
solution mechanism
under floodinginstress,
wheat growth by the application
a gel-free/label-free ofwas
proteomics the
plant-derived smoke solution under flooding stress, a gel-free/label-free proteomics
conducted (Table S1). Three kinds of treatments, which were control, flood, and flood + was
conducted (Table S1). Three kinds of treatments, which were control, flood, and flood +
 Totally, 5774 proteins were identified by LC-MS/MS analysis (Figure 3). The proteo-
mic results of all 9 samples from different 3 groups were compared by principal compo-
nent analysis (PCA), which showed the different accumulation patterns of proteins from
three different kinds of treatment (Figure 3). This result indicated that flooding stress
Plants 2022, 11, 1508
largely affected the wheat proteins; however, this effect was recovered at the protein level 4 of 18
by the application of the plant-derived smoke solution, even if it was under flooding (Fig-
ure 3).

Figure 3. AFigure
Venn diagram
3. A Vennofdiagram
the proteomic results andresults
of the proteomic an overview
and anof the proteomic
overview data of wheat
of the proteomic data of wheat
based on PCA. Wheat seeds were sown and treated with or without the plant-derived smoke solu-
based on PCA. Wheat seeds were sown and treated with or without the plant-derived smoke solution.
tion. Three-day-old wheats were exposed with or without flooding for 3 days. Wheat leaves were
Three-day-old wheats were exposed with or without flooding for 3 days. Wheat leaves were collected
collected for protein extraction. Proteomic analysis was performed with 3 independent biological
foreach
replicates for protein extraction.
treatment. The Proteomic
number inanalysis
the Vennwas performed
diagram showswith 3 independent
the number biological
of proteins iden- replicates
for each treatment. The number in the Venn diagram shows the number of proteins
tified by proteomic analysis. PCA was performed with Proteome Discoverer 2.2 using proteins from identified by
proteomic analysis. PCA was performed with Proteome Discoverer 2.2 using proteins from 9 kinds
9 kinds of samples.
of samples.
The abundance of 314 proteins differentially changed with the p-value < 0.05 and fold
change >1.5 and The<2/3
abundance
in wheatofleaves
314 proteins
under differentially changed
flooding compared to with the p-value
the control < 0.05 and fold
condition
(Table S2).change
Among >1.5
theand <2/3 in wheat
314 proteins, 173 andleaves under flooding
141 proteins increasedcompared to the control
and decreased, respec-condition
tively, under flooding stress compared to the control condition (Table S2 and Figure 4 respec-
(Table S2). Among the 314 proteins, 173 and 141 proteins increased and decreased,
tively,
left). On the otherunder
hand,flooding stress compared
the abundance of anotherto349
theproteins
control condition (Table S2changed
also differentially and Figure 4 left).
On the other hand, the abundance of another 349 proteins also differentially
with the p-value < 0.05 and fold change >1.5 and <2/3 in wheat leaves applied the plant- changed with
the p-value
derived smoke solution under flooding compared to the control condition (Table plant-derived
< 0.05 and fold change >1.5 and <2/3 in wheat leaves applied the S3).
Among thesesmoke349solution under
proteins, flooding
169 and compared
180 proteins to the control
increased condition
and decreased, (Table S3). Among
respectively,
these 349 proteins,
with the application 169 and 180 smoke
of the plant-derived proteins increased
solution underand decreased,
flooding respectively,
compared to the with the
application of the plant-derived smoke solution under flooding compared to the control
control condition (Table S3 and Figure 4 right). The functional category of identified
condition (Table S3 and Figure 4 right). The functional category of identified proteins
was obtained using MapMan bin codes (Figure 4). The abundance of proteins related to
photosynthesis, glycolysis, and amino-acid metabolism was oppositely changed between
the flood/control and flood + smoke/control. To confirm the results obtained from the
proteomic analysis, oppositely changed functional categories, which are photosynthesis,
glycolysis, and amino-acid metabolism, were further analyzed using immunoblot and
amino-acid analyses.
 proteins was obtained using MapMan bin codes (Figure 4). The abundance of proteins
related to photosynthesis, glycolysis, and amino-acid metabolism was oppositely changed
between the flood/control and flood + smoke/control. To confirm the results obtained from
Plants 2022, 11, 1508
the proteomic analysis, oppositely changed functional categories, which are photosynthe-5 of 18
sis, glycolysis, and amino-acid metabolism, were further analyzed using immunoblot and
amino-acid analyses.

Figure 4. The
Figure functional
4. The categories
functional of proteins
categories withwith
of proteins differential abundance
differential in wheat
abundance treated
in wheat withwith
treated
the plant-derived smoke solution under flooding stress. Wheat seeds were sown and treated
the plant-derived smoke solution under flooding stress. Wheat seeds were sown and treated with with
or without the plant-derived smoke solution. Three-day-old wheats were exposed with or without
or without the plant-derived smoke solution. Three-day-old wheats were exposed with or without
flooding. After proteomic analysis, the functional categories of the significantly changed proteins (p
flooding. After proteomic analysis, the functional categories of the significantly changed proteins
< 0.05) from wheat treated with and without the plant-derived smoke solution under flooding were
(p < 0.05)
determined fromMapMan
using wheat treated with(Tables
bin codes and without
S2 and the
S3).plant-derived
Red and blue smoke
columnssolution
show theunder flooding
number
were determined
of increased usingproteins,
and decreased MapMan bin codes (Tables
respectively. S2 and S3).
Abbreviations: AA, Red
aminoand bluemitoETC,
acids; columns mito-
show the
number
chondrial of increased
electron andchain;
transport decreased
OPP,proteins, respectively.
oxidative Abbreviations:
pentose phosphate; AA, amino
and TCA, acids; mitoETC,
tricarboxylic acid
cycle; “not assigned”
mitochondrial indicates
electron proteins
transport without
chain; OPP,ontology
oxidativeorpentose
characterized functions.
phosphate; and TCA, tricarboxylic
acid cycle; “not assigned” indicates proteins without ontology or characterized functions.
2.3. Immunoblot Analysis of Proteins Related to Photosynthesis in Wheat Treated with Plant-
2.3. Smoke
Derived Immunoblot Analysis
Solution under of ProteinsStress
Flooding Related to Photosynthesis in Wheat Treated with
Plant-Derived Smoke Solution under Flooding Stress
As proteins related to photosynthesis were altered in wheat with the application of
As proteins related to photosynthesis were altered in wheat with the application of
the plant-derived smoke solution under flooding stress, the abundance of the ribulose-
the plant-derived smoke solution under flooding stress, the abundance of the ribulose-
bisphosphate carboxylase/oxygenase (RuBisCO) activase, the RuBisCO large subunit, and
bisphosphate carboxylase/oxygenase (RuBisCO) activase, the RuBisCO large subunit, and
the RuBisCO small subunit was selectively analyzed using immunoblot analysis (Figure
the RuBisCO small subunit was selectively analyzed using immunoblot analysis (Figure 5).
5). Proteins
Proteinsextracted
extractedfromfromwheat
wheatleaves
leaveswerewereseparated
separatedon onthe
theSDS-polyacrylamide
SDS-polyacrylamidegel gel by
by electrophoresis
electrophoresisand andtransferred
transferredonto
ontomembranes.
membranes. TheThe
membranes
membranes were cross-reacted
were cross-reacted
withwith
anti-RuBisCO
anti-RuBisCO activase, the RuBisCO
activase, the RuBisCO largelarge
subunit, andand
subunit, the RuBisCO
the RuBisCO small subunit
small subunit
antibodies. A staining pattern with Coomassie-brilliant blue was used as
antibodies. A staining pattern with Coomassie-brilliant blue was used as a loading a loading control
control
(Figure S1). S1).
(Figure The The
integrated densities
integrated of bands
densities werewere
of bands calculated using
calculated ImageJ
using software
ImageJ withwith
software
triplicated immunoblot
triplicated immunoblot results (Figure
results (FigureS2). S2).
The The
abundance of the
abundance of RuBisCO
the RuBisCO activase, the the
activase,
RuBisCO
RuBisCOlargelarge
subunit, and and
subunit, the RuBisCO
the RuBisCO small subunit
small decreased
subunit under
decreased flooding
under stress;
flooding stress;
however, they recovered with the application of the plant-derived smoke
however, they recovered with the application of the plant-derived smoke solution under solution under
flooding (Figure
flooding (Figure5). These results
5). These indicated
results that that
indicated photosynthesis was was
photosynthesis improved
improved by the
by the
plant-derived smoke solution, even if it was under flooding
plant-derived smoke solution, even if it was under flooding conditions.conditions.

2.4. Chlorophyll Contents in Wheat Treated with Plant-Derived Smoke Solution under
Flooding Stress
Using proteomic analysis, because proteins related to photosynthesis were altered in
wheat with the application of the plant-derived smoke solution under flooding stress, the
chlorophyll contents were analyzed as photosynthesis parameters (Figure 6). The contents
of chlorophylls a and b significantly decreased by flooding stress; however, they were
recovered by the application of the plant-derived smoke solution (Figure 6). These results
 Plants 2022, 11, 1508 6 of 18

, 11, x FOR PEER REVIEW 6 of 18

indicated that photosynthesis was improved by the plant-derived smoke solution, even if
it was under flooding conditions.

Figure 5. ImmunoblotFigure analysis

5. of the proteins analysis
Immunoblot involved ofin photosynthesis in wheatin
the proteins involved treated with the in wheat treated with
photosynthesis
plant-derived smoke solution under flooding stress. Proteins extracted from leaves of wheat seed-
the plant-derived smoke solution under flooding stress. Proteins extracted from leaves of wheat
lings were separated on SDS-polyacrylamide gel by electrophoresis and transferred onto mem-
branes. The membranes seedlings were separated
were cross-reacted on SDS-polyacrylamide
with anti-RuBisCO activase, thegel by electrophoresis
RuBisCO large subunit,and transferred onto mem-
and the RuBisCO small subunit antibodies. A staining pattern with Coomassie-brilliant blue was RuBisCO large subunit,
branes. The membranes were cross-reacted with anti-RuBisCO activase, the
used as a loading control (Figure
and the S1). The
RuBisCO integrated
small subunit densities of theAbands
antibodies. were
staining calculated
pattern withusing
Coomassie-brilliant blue was
ImageJ software. Theused data as
area presented as mean ± SD from 3 independent biological replicates
loading control (Figure S1). The integrated densities of the bands were calculated using
(Figure S2). Asterisks ImageJ
indicatesoftware.
significantThe
changes
datainare
thepresented
relative intensity
as meanof signal
± SD band
fromin3 the plant-
independent biological replicates
derived smoke solution under flooding compared to only flooding according to the Student’s t-test
(Figure S2). Asterisks indicate significant changes in the relative intensity of signal band in the
11, x FOR PEER REVIEW(**, p < 0.01; *, p < 0.05). 7 of 18
plant-derived smoke solution under flooding compared to only flooding according to the Student’s
2.4. Chlorophyll Contents t-test in p < 0.01;
(**,Wheat *, p <with
Treated 0.05).
Plant-Derived Smoke Solution under Flooding
Stress
Using proteomic analysis, because proteins related to photosynthesis were altered in
wheat with the application of the plant-derived smoke solution under flooding stress, the
chlorophyll contents were analyzed as photosynthesis parameters (Figure 6). The contents
of chlorophylls a and b significantly decreased by flooding stress; however, they were re-
covered by the application of the plant-derived smoke solution (Figure 6). These results
indicated that photosynthesis was improved by the plant-derived smoke solution, even if
it was under flooding conditions.

Figure 6. The contentsFigure 6. The contents

of chlorophylls a and bofinchlorophylls
wheat treated a and
withb the
in wheat treated with
plant-derived smokethesolu-
plant-derived smoke solution
tion under flooding stress.
underChlorophylls a and Chlorophylls
flooding stress. b extracted from the leaves
a and of wheat
b extracted seedlings
from were of wheat seedlings were
the leaves
measured. Asterisks indicate
measured. significant
Asteriskschanges in the
indicate relative intensity
significant changes ofin the
thesignal band
relative in the of the signal band in the
intensity
plant-derived smoke solution under flooding compared to only flooding according to the Student’s
plant-derived smoke solution under flooding compared to only flooding according to the Student’s
t-test (**, p < 0.01).
t-test (**, p < 0.01).
2.5. Immunoblot Analysis of Proteins Related to Glycolysis in Wheat Treated with Plant-Derived
Smoke Solution under Flooding Stress
As proteins related to glycolysis were altered in wheat with the application of the
plant-derived smoke solution under flooding stress, the abundance of fructose-bisphos-
 Plants 2022, 11, 1508 7 of 18

2.5. Immunoblot Analysis of Proteins Related to Glycolysis in Wheat Treated with Plant-Derived
Smoke Solution under Flooding Stress
As proteins related to glycolysis were altered in wheat with the application of the plant-
derived smoke solution under flooding stress, the abundance of fructose-bisphosphate al-
dolase (FBPA), triose-phosphate isomerase (TPI), and glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) was selectively analyzed using the immunoblot analysis (Figure 7). Pro-
teins extracted from the leaves and roots of wheat were separated on SDS-polyacrylamide
gel by electrophoresis and transferred onto membranes. The membranes were cross-reacted
with anti-FBPA, TPI, and GAPDH antibodies. A staining pattern with Coomassie-brilliant
blue was used as a loading control (Figure S1). The integrated densities of bands were
calculated using ImageJ software with triplicated immunoblot results (Figures S3–S5). The
abundance of FBPA increased by flooding stress; however, it was recovered in wheat leaves
with the application of the plant-derived smoke solution (Figure 7). The abundance of
GAPDH decreased by flooding stress, and it was further decreased in wheat leaves with the
application of the plant-derived smoke solution (Figure 7). On the other hand, TPI did not
1, x FOR PEER REVIEW change with the application of the plant-derived smoke solution8 (Figure
of 18 7). These results
indicated that the balance of glycolysis-related proteins, which were FBPA and GAPDH,
was affected by the plant-derived smoke solution.

Figure 7. ImmunoblotFigure
analysis7. ofImmunoblot
the proteins analysis
involvedof in the
glycolysis in wheat
proteins treated
involved with the plant-
in glycolysis in wheat treated with the
derived smoke solution under flooding stress. Proteins extracted from the leaves and roots of wheat
plant-derived smoke solution under flooding stress. Proteins extracted from the leaves and roots
seedlings were separated on SDS-polyacrylamide gel by electrophoresis and transferred onto mem-
of wheat seedlings were separated on SDS-polyacrylamide gel by electrophoresis and transferred
branes. The membranes were cross-reacted with anti-FBPA, TPI, and GAPDH antibodies. A staining
onto membranes.
pattern with Coomassie-brilliant Theused
blue was membranes
as a loadingwere cross-reacted
control (Figure with anti-FBPA,
S1). The TPI,
integrated and GAPDH antibodies.
den-
A staining pattern with Coomassie-brilliant blue was used as
sities of bands were calculated using ImageJ software. The data are presented as mean ± SD from 3 a loading control (Figure S1). The
integrated densities of bands were calculated using ImageJ software.
independent biological replicates (Figures S3–S5). Asterisks indicate significant changes in the rela- The data are presented as
mean ± SD from 3 independent biological replicates (Figures S3–S5). Asterisks indicate significant
tive intensity of the signal band in the plant-derived smoke solution under flooding compared to
only flooding according to theinStudent’s
changes t-test
the relative (**: p <of0.01).
intensity the signal band in the plant-derived smoke solution under flooding
compared to only flooding according to the Student’s t-test (**: p < 0.01).
2.6. Immunoblot Analysis of Proteins Related to Biotic Stress in Wheat Treated with Plant-De-
rived Smoke Solution under Flooding Stress
Using proteomic analysis, the abundance of pathogen-related protein (PR)-1 and PR
10 increased under flooding stress (Table S2) and decreased with the application of the
plant-derived smoke solution (Table S3) in wheat leaves. On the other hand, thaumatin,
 Plants 2022, 11, 1508 8 of 18

2.6. Immunoblot Analysis of Proteins Related to Biotic Stress in Wheat Treated with Plant-Derived
Smoke Solution under Flooding Stress
Using proteomic analysis, the abundance of pathogen-related protein (PR)-1 and PR
10 increased under flooding stress (Table S2) and decreased with the application of the
plant-derived smoke solution (Table S3) in wheat leaves. On the other hand, thaumatin,
named PR5, mildly increased under flooding stress and significantly increased by the
application of the plant-derived smoke solution under flooding stress (Tables S2 and S3).
As proteins related to biotic stress were altered in wheat with the application of the plant-
derived smoke solution under flooding stress, the abundance of PR1, PR5, and PR10 was
selectively analyzed using immunoblot analysis (Figure 8). Proteins extracted from wheat
leaves were separated on SDS-polyacrylamide gel by electrophoresis and transferred onto
membranes. The membranes were cross-reacted with anti-PR1, PR5, and PR10 antibodies.
A staining pattern with Coomassie-brilliant blue was used as a loading control (Figure S1).
The integrated densities of the bands were calculated using ImageJ software with triplicated
immunoblot results. The abundance of the PR1, PR5, and PR10 increased under flooding
stress; however, it was recovered with the application of the plant-derived smoke solution
2, 11, x FOR PEER REVIEW
under flooding (Figure 8). These results indicated that9 biotic
of 18
stress was suppressed by the
plant-derived smoke solution, even if it was under flooding conditions.

Figure 8. Immunoblot analysis

Figure of8. the proteins involved
Immunoblot analysisinof
biotic stress in wheat
the proteins treated
involved with the
in biotic stress in wheat treated with the plant-
plant-derived smoke solution under flooding stress. Proteins extracted from the leaves of wheat
seedlings were separated on SDS-polyacrylamide gel by electrophoresis and transferred onto mem-from the leaves of wheat seedlings
derived smoke solution under flooding stress. Proteins extracted
branes. The membraneswere were separated
cross-reactedonwith SDS-polyacrylamide
anti-PR1, PR5, and PR10 gel by electrophoresis
antibodies. A stainingand transferred onto membranes.
pattern with Coomassie-brilliant
The membranes were cross-reacted with anti-PR1, PR5, andden-
blue was used as a loading control (Figure S1). The integrated PR10 antibodies. A staining pattern
sities of the bands were calculated using ImageJ software. The data are presented as mean ± SD from
with Coomassie-brilliant blue was used as a loading control (Figure S1). The integrated densities
3 independent biological replicates (Figures S3–S5). Asterisks indicate significant changes in the rel-
of the
ative intensity of signal band in bands were calculated
the plant-derived using under
smoke solution ImageJ software.
flooding The to
compared onlyare presented as mean ± SD from
data
flooding according to the3 Student’s
independent biological
t-test (**: p < 0.01). replicates (Figures S3–S5). Asterisks indicate significant changes in the
relative intensity of signal band in the plant-derived smoke solution under flooding compared to
2.7. Amino-Acid Analysis
onlyin Wheat Treated
flooding with Plant-Derived
according Smoke
to the Student’s Solution
t-test (**: punder Flooding
< 0.01).
Stress
As proteins related to amino-acid metabolism were altered in wheat with the treat-
ment of the plant-derived smoke solution under flooding stress, the abundance of amino
acids was analyzed using the automatic amino-acid analyzer. In total, 32 amino acids were
identified in wheat (Table S4) and mapped on amino-acid metabolism using the KEGG
database (Figure 9). In altered amino acids, the abundance of glutamine (Gln), glutamic
 Plants 2022, 11, 1508 9 of 18

2.7. Amino-Acid Analysis in Wheat Treated with Plant-Derived Smoke Solution under
Flooding Stress
As proteins related to amino-acid metabolism were altered in wheat with the treatment
of the plant-derived smoke solution under flooding stress, the abundance of amino acids
was analyzed using the automatic amino-acid analyzer. In total, 32 amino acids were
identified in wheat (Table S4) and mapped on amino-acid metabolism using the KEGG
database (Figure 9). In altered amino acids, the abundance of glutamine (Gln), glutamic
acid (Glu), aspartic acid (Asp), and serine (Ser) decreased under flooding; however, it
was recovered by the application of the plant-derived smoke solution. The abundance
of ornithine and phenylalanine (Phe) significantly increased under flooding; however, it
was recovered by the application of the plant-derived smoke solution. The abundance of
alanine (Ala), citrulline, valine (Val), and gamma-aminobutyric acid (GABA) increased
under flooding; it further increased with the application of the plant-derived smoke solution
Plants 2022, 11, x FOR PEER REVIEW (Figure 9). These results indicated that amino-acid metabolism was significantly 10 ofaffected
18

by the plant-derived smoke solution under flooding.

Figure 9. A 9.
Figure mapping
A mappingof altered amino
of altered acids
amino to to
acids amino-acid
amino-acidmetabolism
metabolismin in wheat treated with
wheat treated withthe
theplant-
plant-derived smoke solution under flooding stress. Totally, 32 amino acids identified using an au-
derived smoke solution under flooding stress. Totally, 32 amino acids identified using an automatic
tomatic amino-acid analyzer were mapped onto pathways according to the KEGG database. Amino-
amino-acid analyzer were mapped onto pathways according to the KEGG database. Amino-acids
acids analysis was performed with 3 independent biological replicates for each treatment (Table S4).
analysis was
The different performed
colors indicate with 3 independent
the different biological
ratio ranges replicates
of the for of
quantities each treatment which
metabolites, (Table S4).
are The
different colors indicate the different ratio ranges of the quantities of metabolites,
calculated using the contents of wheat treated with or without the plant-derived smoke solution which are calculated
under using the contents
flooding by thoseoffrom
wheat treated with
untreated wheat.or Each
without
set the
of 2plant-derived smoke
boxes shows that thesolution under flooding
left is “flood/con-
trol” by
andthose
the right is “flood + smoke/control”. Abbreviations: GABA, gamma-aminobutyric
from untreated wheat. Each set of 2 boxes shows that the left is “flood/control” acid.and the
right is “flood + smoke/control”. Abbreviations: GABA, gamma-aminobutyric acid.
3. Discussion
3. Discussion
3.1. Plant-Derived Smoke Solution Improves Flooding Tolerance of Wheat
3.1. Plant-Derived Smoke Solution Improves Flooding Tolerance of Wheat
The use of organic fertilizers and plant-derived herbicides for seed or plant treatment
The use of organic fertilizers and plant-derived herbicides for seed or plant treatment
is the sensible example of efforts in the direction of sustainable agricultural practices. This
is the sensible example of efforts in the direction of sustainable agricultural practices.
result shows an increasing demand for such naturally derived agro-chemicals for sustain-
This result shows an increasing demand for such naturally derived agro-chemicals for
able farming systems. It was reported that root/shoot length, fresh weight, and dry weight,
sustainable farming systems. It was reported that root/shoot length, fresh weight, and dry
as well as leaf area, were improved by the plant-derived smoke solution in wheat under
weight, as well as leaf area, were improved by the plant-derived smoke solution in wheat
optimum conditions [16]. However, in the present study, the leaf length/weight and root
length/weight of wheat were not improved by the plant-derived smoke solution under
optimum conditions (Figure 2). It might be affected by the differences of the materials of
smoke solution between Cymbopogon jwarncusa used in this study and plants used in the
previous study, which were rice, Cynodon dactylon, Pongamia glabra, Populus deltoides, and
Plants 2022, 11, 1508 10 of 18

under optimum conditions [16]. However, in the present study, the leaf length/weight and
root length/weight of wheat were not improved by the plant-derived smoke solution under
optimum conditions (Figure 2). It might be affected by the differences of the materials of
smoke solution between Cymbopogon jwarncusa used in this study and plants used in the
previous study, which were rice, Cynodon dactylon, Pongamia glabra, Populus deltoides, and
Morus alba [16]. In the case of soybean, the plant-derived smoke solution prepared from
Cymbopogon jwarncusa increased the length of the root, including hypocotyl under optimum
conditions, although its weight did not change [13]. Because the effect of the plant-derived
smoke solution is not the same among plant species [6], the effect on plant growth might
be different between soybean and wheat.
Karrikin, which is one component of the plant-derived smoke solution [18], regulated
tolerance to abiotic stresses such as drought [19] and cold [20] in Arabidopsis thaliana as
well as salt [21] and cadmium [22] in oil plant. Furthermore, it was reported that the
plant-derived smoke solution enhanced soybean growth under flooding [13] and after
flooding [12]. In this study, the length and weight of wheat leaves increased by the
application of plant-derived smoke under flooding stress (Figure 2). This result with
previous findings suggests that the plant-derived smoke solution has a positive effect
against abiotic stress, including flooding stress.

3.2. Photosynthesis Activity Increases in Wheat by Plant-Derived Smoke Solution under Flooding
The combined solution of Bacillus safensis and plant-derived smoke prepared from Cym-
bopogon jwarancusa primed seeds increased the germination percentage, seedling growth,
ion contents, and photosynthetic pigments, such as chlorophyll a and chlorophyll b [23].
Most proteins involved by karrikin are related to photosynthesis, carbohydrate metabolism,
redox homeostasis, transcription control, proteosynthesis, and protein metabolism in Ara-
bidopsis thaliana [24]. Li et al. [25] reported that cytokinin and brassinosteroid metabolism
was specifically regulated by the D14, strigolactone receptor dwarf14, pathway, whereas the
photosynthesis and metabolism of glucosinolates and trehalose were potentially regulated
by both D14 and KAI2, karrikin receptor karrikin insensitive 2, pathways in plant response to
water scarcity. In this study, the abundance of RuBisCO activase and RuBisCO large/small
subunits decreased by flooding stress; however, they were recovered in wheat with the
application of the plant-derived smoke solution (Figure 5). Furthermore, the contents of
chlorophyll a and chlorophyll b were also recovered in wheat with the application of the
plant-derived smoke solution (Figure 6). These results with the previous report suggest
that the plant-derived smoke solution improved wheat-leaf growth through photosynthesis
activation, even if it was under flooding.

3.3. Glycolysis Is Suppressed in Wheat by Plant-Derived Smoke Solution under Flooding

Glycolysis and gluconeogenesis were activated to generate energy for soybean-plant
survival under anaerobic conditions [26]. In the case of chickpea, FBPA increased, while
phosphoglycerate mutase decreased in glycolysis by the plant-derived smoke solution un-
der optimum condition [11]. On the other hand, sucrose/starch metabolism and glycolysis
were suppressed in soybean treated with the plant-derived smoke solution under flooding
compared to flooded soybean [12]. In this study, FBPA and GAPDH decreased in wheat
leaves by the application of plant-derived smoke under flooding (Figure 7). FBPA and
GAPDH are located at the key intersections between glycolysis and the pentose-phosphate
pathway, which are required for both pathways and are essential for the synthesis of
glucose [27]. Additionally, both FBPA and GAPDH exert so-called “moonlighting” func-
tions in yeast and other organisms, which are biological activities in addition to their
catalytic role in glycolysis and gluconeogenesis [28]. Plants overcome their oxygen lim-
itations and adapt by reducing alcohol fermentation, which is toxic to plants because of
the production of ethanol, and by enhancing glycolysis. However, plant-derived smoke
suppresses the glycolysis pathway in wheat, which might mildly generate the energy for
surviving long-term under flooding stress.
Plants 2022, 11, 1508 11 of 18

3.4. PR Proteins Are Accumulated in Wheat under Flooding and Suppressed by Plant-Derived
Smoke Solution under Flooding
PR proteins are an integral part of the defense mechanisms of plants against various
types of abiotic and biotic stresses [29]. Plants evolved different kinds of defense mecha-
nisms, including physical and chemical defenses, to protect themselves from pathogens,
which terminate pathogen infection and disease development [30]. Flooding stress limits
the flow of light to plants, induces hypoxia in plants, and increases their vulnerability to
pathogen attacks [31,32]. Interacted proteins with SUB1A, which is the master regulator of
submergence tolerance, improved the crosstalk between submergence stress and pathogen
defense and the modulation of elongation, respectively [33]. In this study, PR proteins
increased under flooding stress and decreased with the application of the plant-derived
smoke solution under flooding stress (Figure 8). The present result with previous reports
indicates that flooding stress increases the vulnerability to pathogen attack and that the
pathogen-defense system is important to recovering from flooding stress.

3.5. Amino Acids Are Accumulated by Flooding and Suppressed by the Application of
Plant-Derived Smoke Solution
The ability of plant-derived smoke to act as a plant growth inducer in many species has
led to widespread interest in plant biology. Karrikin was identified as the main component
of plant-derived smoke formed from the reaction of sugars with amino acids [34]. Flooding
resulted in a marked decrease of asparagine (Asn), which is the most abundant amino
acid, and a concomitant accumulation of GABA [35]. In the present study, the abundance
of Asn and GABA decreased and increased, respectively (Figure 8). The submergence
inhibited photosystem II photochemistry and stimulated the breakdown of protein and
the accumulation of several amino acids in rice. The accumulation of five amino acids
such as arginine (Arg), Phe, proline (Pro), threonine (Thr), and Val was highly elevated
in response to submergence in a submergence-sensitive line [31]. When the plant was
exposed to desubmergence, the amount of each amino acid gradually declined, which
reached the level of non-stress plants more rapidly in a submergence-tolerant line [36].
In this study, Arg, Phe, Thr, and Val also accelerated in wheat by flooding; on the other
hand, additional the plant-derived smoke solution suppressed the accumulation of Arg
(Figure 9). The abundance of Ala and tyrosine (Tyr) were elevated by submergence, but
the accumulation of these amino acids was more abundant in submergence-sensitive line
under the stress [36]. In this study, the abundance of Ala and Tyr increased under flooding
and further increased by the application of plant-derived smoke (Figure 9). This result with
the previous finding suggests that plant-derived smoke contributes to the metabolism of
amino acids for the survival of wheat from flooding.
Asp participates in glycolysis, the conjugation of indole-3-acetic acid/ethylene, and
the cross-talk between salicylic acid/ jasmonic acid, indicating that the primary features
for plant growth and immune control are N recycling, translocation, and signaling [37].
The physiological impacts of Asp in plants are uncovering the conspicuous roles of Asp
in regulating the plant adaptation and tolerance to abiotic and biotic stress cues [38]. The
responsive behavior of the primary metabolism in association with energy processing,
including glycolysis and the pentose-phosphate pathway, ATP, the tricarboxylic acid (TCA)
cycle, and the biosynthesis of amino acids, requires for energy production (Lys and Met)
and photorespiration (Glu, Arg, Ser, and Gly) responding various stress cues [39]. In this
study, Asp significantly decreased under flooding stress and recovered with the application
of the plant-derived smoke solution (Figure 9), suggesting that plant-derived smoke can
rescue wheat from flooding stress.
Glutamate plays a central role in amino acid metabolism, in particular, in aminotrans-
ferase reactions leading to the formation of many other proteinogenic and nonproteinogenic
amino acids. In stress conditions, glutamate can be either metabolized to GABA by glu-
tamate decarboxylase, which initiates a GABA shunt bypassing several reactions of the
TCA cycle, or converted to 2-oxoglutarate by glutamate dehydrogenase [40]. GABA plays a
Plants 2022, 11, 1508 12 of 18

dual role in regulating the C:N balance and nitrogen metabolism, as well as being involved
in many physiological processes, such as carbon flux in TCA cycle and the antioxidant
effect. Furthermore, GABA acts as an important signal that triggers a series of down-
stream responses, such as cold or salt stress tolerance; regulates cytoplasmic pH; and
controls programmed cell death [41,42]. Concurrently, verifying the altered accumulation
of amino acids such as Glu, Asp, Asn, Pro, and GABA prompted the potential to be a
defense indicator, aiding in uncovering the synergistically fine-tuned Asp pathway upon
flooding stress.

4. Materials and Methods

4.1. Plant Material and Treatment
The plant-derived smoke solution was prepared from semi-dried Cymbopogon jwarn-
cusa (Kohat University of Science and Technology, Kohat, Pakistan) [13], which was
modified from previous methods [43]. The seeds of wheat (Triticum aestivum L. cultivar
Nourin 61; Asahi Noen Seed, Inasawa, Japan) were sterilized with 2% sodium hypochlorite
solution, rinsed with water, and sown with or without 2000 ppm plant-derived smoke in
400 mL of silica sand in a seedling case. Plants were grown in a growth chamber with
white fluorescent light (16 h light of 200 µmol m−2 s−1 and 8 h dark photoperiod) with
60% humidity at 25 ◦ C. Three-day-old plants were flooded for three days. Leaf length,
leaf-fresh weight, main-root length, and total-root fresh weight were measured 6 days after
sowing. Three independent experiments were performed as biological replicates for all
experiments. In each experiment, 20 seeds were sown for each replication of each treat-
ment. For the morphological experiment, 10 seedlings were collected for each replication
of each treatment. For other biological experiments, 5–10 seedlings were collected for each
replication of each treatment. The sowing of seeds was carried out on different days for
making biological replicates.

4.2. Protein Extraction

A portion (300 mg) of leaves of wheat was excised into small pieces and put into a
filter cartridge (Cosmo Bio, Carlsbad, CA, USA). It was ground with a plastic rod 120 times
in 75 µL of lysis buffer, which contained 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM
tributylphosphine. The suspension was incubated for 2 min at 25 ◦ C and centrifuged twice
with 15,000× g at 4 ◦ C for 5 min. The detergents from the supernatant were removed
using the Pierce Detergent Removal Spin Column (Pierce Biotechnology, Rockford, IL,
USA). The protein concentration was determined with the Bradford method [44] with the
bovine serum albumin as the standard. Quantified proteins were used for proteomic and
immunoblot analyses.

4.3. Protein Enrichment, Reduction, Alkylation, and Digestion

Extracted proteins (100 µg) were adjusted to a final volume of 100 µL. To each sample
was added 400 µL of methanol, and it was mixed before the addition of 100 µL of chloroform
and 300 µL of water. After centrifugation at 20,000× g for 10 min, the upper phase was
discarded and 300 µL of methanol was added to the lower phase. After centrifugation
at 20,000× g for 10 min, the pellet was resuspended in 50 mM NH4 HCO3 , reduced with
50 mM dithiothreitol for 30 min at 56 ◦ C, and alkylated with 50 mM iodoacetamide for
30 min at 37 ◦ C in the dark. Alkylated proteins were digested with trypsin and lysyl
endopeptidase (Wako, Osaka, Japan) at a 1:100 enzyme/protein ratio for 16 h at 37 ◦ C.
Peptides were desalted with MonoSpin C18 Column (GL Sciences, Tokyo, Japan) and
acidified with 1% trifluoroacetic acid [45].

4.4. Protein Identification Using LC-MS/MS

Peptides were analyzed by LC (EASY-nLC 1000; Thermo Fisher Scientific, San Jose,
CA, USA) combined with MS/MS (Orbitrap Fusion ETD MS; Thermo Fisher Scientific, San
Jose, CA, USA) as described in the previous study [46] (Table S1). The peptides were loaded
Plants 2022, 11, 1508 13 of 18

onto the LC system equipped with a trap column (Acclaim PepMap 100 C18 LC column,
3 µm, 75 µm ID × 20 mm; Thermo Fisher Scientific, San Jose, CA, USA) equilibrated with
0.1% formic acid and eluted with a linear acetonitrile gradient (0–35%) in 0.1% formic
acid at a flow rate of 300 nL/min. The eluted peptides were loaded and separated on the
column (EASY-Spray C18 LC column, 3 µm, 75 µm ID x 150 mm; Thermo Fisher Scientific,
San Jose, CA, USA) with a spray voltage of 2 kV (Ion Transfer Tube temperature: 275 ◦ C).
The peptide ions were detected using MS in the data-dependent acquisition mode with
the installed Xcalibur software (version 4.0; Thermo Fisher Scientific, San Jose, CA, USA).
Full-scan mass spectra were acquired in the MS over 375–1500 m/z with a resolution of
120,000. The most intense precursor ions were selected for collision-induced fragmentation
in the linear ion trap at a normalized collision energy of 35%. Dynamic exclusion was
employed within 60 sec to prevent the repetitive selection of peptides.

4.5. Analysis of MS/MS Data

The MS/MS searches were carried out using MASCOT (version 2.6.1, Matrix Science,
London, UK) and SEQUEST HT search algorithms against the UniProtKB Triticum aes-
tivum protein database (25 October 2017) using Proteome Discoverer 2.2 (version 2.2.0.388;
Thermo Scientific, San Jose, CA, USA). The condition of analysis is described in the previ-
ous study [13] (Table S1). The workflow for both algorithms included spectrum files RC,
spectrum selector, MASCOT, SEQUEST HT search nodes, percolator, ptmRS, and minor
feature detector nodes. The oxidation of methionine was set as a variable modification, and
the carbamidomethylation of cysteine was set as a fixed modification. MS and MS/MS
mass tolerances were set to 10 ppm and 0.6 Da, respectively. Trypsin was specified as
protease, and a maximum of one missed cleavage was allowed. Target-decoy database
searches were used for the calculation of the false discovery rate, which was set at 1% for
peptide identification.

4.6. Differential Analysis of Proteins Using MS Data

Label-free quantification was performed with Proteome Discoverer 2.2 using precursor
ions quantifiler nodes. Principal component analysis (PCA) was also performed with Pro-
teome Discoverer 2.2. For the differential analysis of the relative abundance of peptides and
proteins between samples, the freely software Perseus (version 1.6.2.3, Max Planck Institute
of biochemistry, Martinsried, Germany) was used. The condition of analysis is described in
the previous study [47]. Proteins and peptides abundances were transferred into the log2
scale. Three biological replicates of each sample were grouped, and a minimum of three
valid values were required in at least one group. The normalization of the abundances was
performed to subtract the median of each sample. Missing values were imputed based on a
normal distribution (width = 0.3, down-shift = 1.8). The significance was assessed using
t-test analysis.

4.7. Immunoblot Analysis

Proteins extracted from leaves and roots were added in an SDS-sample buffer con-
sisting of 60 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 50 mM dithiothreitol
as the final concentration [48]. Proteins (10 µg) were separated by electrophoresis on a
10% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane
using a semidry transfer blotter (Nippon Eido, Tokyo, Japan). The blotted membrane
was blocked for 5 min in Bullet Blocking One reagent (Nacalai Tesque, Kyoto, Japan).
After blocking, the membrane was cross-reacted with a 1:1000 dilution of the primary
antibodies for 30 min. As the primary antibodies, the followings were used: anti-ribulose
bisphosphate carboxylase/oxygenase (RuBisCO) activase [49]; RuBisCO large subunit [50];
RuBisCO small subunit [50]; fructose-bisphosphate aldolase (FBPA) [51]; triose-phosphate
isomerase (TPI) [52]; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [52]; and the
pathogen-related protein (PR) 1 [53], PR5 [53], and PR10 [53] antibodies. As the secondary
antibody, anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA,
Plants 2022, 11, 1508 14 of 18

USA) was used for 30 min incubation. The signals were detected using the TMB Membrane
Peroxidase Substrate kit (Seracare, Milford, MA, USA). Coomassie brilliant blue staining
was used as a loading control. The integrated densities of bands were calculated using
Image J software (version 1.53e with Java 1.8.0_172; National Institutes of Health, Bethesda,
MD, USA).

4.8. Contents of Chlorophylls a and b

A portion (500 mg) of leaves of wheat was submerged in 1 mL of in N,N-dimethylformamide
for 16 h at 4 ◦ C. The absorbance of chlorophylls a and b released in the solvent was measured
at 663.8 nm and 646.8 nm. Using absorbance, the contents of chlorophylls a and b were
calculated as follows: chlorophylls a and b (µM) = 19.4 × A646.8 + 8.05 ×A663.8 [54].

4.9. Amino-Acid Analysis

A portion (500 mg) of leaves of wheat was ground in phosphate-buffered saline, in-
cluding 140 mM NaCl, 2.7 mM KCl, and 10 mM PO4 3-, using a mortar and pestle. The
suspension was centrifuged at 20,000× g for 20 min at 4 ◦ C, and the supernatant was
re-centrifuged with the same condition. The final supernatant was mixed with the same
amount of 3% sulfosalicylic acid and centrifuged 20,000× g for 20 min at 4 ◦ C to remove
precipitated proteins. After filtration, the amino-acid concentrations in supernatants ob-
tained were analyzed with ninhydrin regent using a fully automatic amino-acid analyzer
(JLC-500/V; JEOL, Tokyo, Japan).

4.10. Statistical Analysis, Gene Annotation, and Metabolite Mapping

The statistical significance of the data was analyzed by the Student’s t-test. A p-value
of less than 0.05 was considered statistically significant. The gene functional annotations
and protein categorization were analyzed using MapMan bin codes [55]. Amino acids
were mapped using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database
(https://www.genome.jp/kegg/mapper.html; 4 June 2022).

5. Conclusions
Wheat is a highly adaptable food crop that is grown extensively around the world;
however, its growth is reduced by flooding. Currently, it was reported that the plant-
derived smoke solution enhances soybean growth under flooding [12,13]; however, its
growth-promoting mechanism is not clearly understood. The present study identified
that the plant-derived smoke solution improved the wheat growth, even if it was under
flooding. To reveal the role of the plant-derived smoke solution in wheat under flood-
ing, a gel-free/label-free proteomic analysis was conducted and the results were further
confirmed using biochemical techniques (Figure 10). The main findings are as follows:
(i) according to a functional categorization, oppositely changed proteins were correlated
with photosynthesis, glycolysis, biotic stress, and amino-acid metabolism between with
and without the plant-derived smoke solution under flooding.; (ii) immunoblot analysis
confirmed that RuBisCO activase and RuBisCO large/small subunits decreased in leaves
under flooding and recovered by the application of the plant-derived smoke solution;
(iii) in glycolysis-related proteins, FBPA and GAPDH decreased by the application of the
plant-derived smoke solution under flooding compared with flooding alone.; (iv) PR1
and PR10 increased under flooding stress and recovered by the application of the plant-
derived smoke solution., and (v) amino-acid analysis confirmed that Gln, Glu, Asp, and
Ser decreased by flooding and recovered by the plant-derived smoke solution. These
results suggest that the application of plant-derived smoke to wheat improves the recovery
of plant growth through the regulation of photosynthesis, and glycolysis. Furthermore,
plant-derived smoke might promote wheat tolerance against flooding and biotic stresses
through the regulation of amino-acid metabolism.
 Plants
22, 11, x FOR PEER2022, 11, 1508
REVIEW 16 of 18 15 of 18

Figure 10. The overall

Figureresponses of theresponses
10. The overall main proteins
of thein the proteins
main functional
in categories in wheat
the functional leaf to
categories in wheat leaf to the
the plant-derivedplant-derived
smoke solution under
smoke flooding
solution stress.
under flooding stress.

Supplementary Supplementary
Materials: The Materials:
following The supporting
followinginformation can be downloaded
supporting information at:
can be downloaded at: https:
www.mdpi.com/xxx/s1, Table S1. The experimental procedure of gel-free/label-free
//www.mdpi.com/article/10.3390/plants11111508/s1, Table S1. Theproteomics
experimental procedure of
used in this research [58–60]. Table S2.
gel-free/label-free A list of changed
proteomics used in proteins in wheat
this research leaves Table
[12,46,47]. treated with
S2. flood-
A list of changed proteins
ing compared with control. Table S3. A list of changed proteins in wheat leaves treated with flood-
in wheat leaves treated with flooding compared with control. Table S3. A list of changed proteins
ing and the plant-derived smoke solution compared with control. Table S4. A list of amino acids in
in wheat leaves treated with flooding and the plant-derived smoke solution compared with control.
wheat leaves treated with or without the plant-derived smoke solution under flooding compared
Table S4. A list of amino acids in wheat leaves treated with or without the plant-derived smoke
with control. Figure S1. The Coomassie brilliant blue staining patterns of proteins used for immuno-
solution under flooding compared with control. Figure S1. The Coomassie brilliant blue staining
blot analysis. Figure S2. Blots of the entire membrane with anti-RuBisCO activase, the RuBisCO
patterns of proteins used for immuno-blot analysis. Figure S2. Blots of the entire membrane with
large subunit, and the RuBisCO small subunit antibodies, which are used in Figure 5. Figure S3.
Blots of the entireanti-RuBisCO
membrane with activase, the RuBisCO
anti-FBPA antibody,large subunit,
which and the
are used RuBisCO
in Figure small S4.
7. Figure subunit
Blotsantibodies, which
are used in Figure 5. Figure S3. Blots of the entire membrane with anti-FBPA
of the entire membrane with anti-TPI antibody, which are used in Figure 7. Figure S5. Blots of the antibody, which are
used in Figure 7. Figure S4. Blots of the entire
entire membrane with anti-GAPDH antibody, which are used in Figure 7. membrane with anti-TPI antibody, which are used
in Figure 7. Figure S5. Blots of the entire membrane with anti-GAPDH antibody, which are used
Author Contributions: Conceptualization,
in Figure 7. S.K.; smoke-solution preparation, S.U.R.; sample prepa-
ration, S.K.; amino-acid analysis. T.O.; MS analysis, H.Y., K.H., and K.T.; biological experiments and
Author
data analyses, S.K.; review, Conceptualization,
Contributions:
and writing, S.K.;
and editing, S.K.; all smoke-solution
authors have readpreparation,
and agreed S.U.R.;
to the sample prepara-
published versiontion, S.K.;
of the amino-acid analysis. T.O.; MS analysis, H.Y., K.H. and K.T.; biological experiments and
manuscript.
data analyses, S.K.; and writing, review, and editing, S.K.; All authors have read and agreed to the
Funding: This research received
published no of
version external funding.
the manuscript.
Data AvailabilityFunding:
Statement: For
This MS data,
research RAW no
received data, peak lists,
external and result files have been depos-
funding.
ited in the ProteomeXchange Consortium [56] via the jPOST [57] partner repository under data-set
Data Availability Statement: For MS data, RAW data, peak lists, and result files have been deposited
identifiers PXD017690.
in the ProteomeXchange Consortium [56] via the jPOST [57] partner repository under data-set
Conflicts of Interest: The authors
identifiers declare no conflict of interest.
PXD017690.

nces Conflicts of Interest: The authors declare no conflict of interest.

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