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ISSN 1021-4437, Russian Journal of Plant Physiology, 2024, Vol. 71:2. © Pleiades Publishing, Ltd., 2024.
Genome-Wide Identification of Ascorbate Peroxidase Gene Family
in Two Contrasting Barley (Hordeum vulgare L.) Cultivars
in Response to Abiotic Stresses
A. Saidia, *, Z. Hajibarata, and H. Ghazvinib
a Department of Cell and molecular biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University,
Tehr a n, Ira n
b Cereal Research Department, Seed and Plant Improvement Institute, Agricultural Research, Education,
and Extension Organization (AREEO), Karaj, Iran
*e-mail: abbas.saidi@gmail.com
Received September 18, 2023; revised December 2, 2023; accepted December 5, 2023
Abstract—Reactive oxygen species, as main molecules, participate in the adaptation process of plants under
environmental stimuli. Excessive oxidation and reduction of cellular components are equally harmful, so
maintaining redox homeostasis is critical. The comprehensive analysis of HvAPX genes in barley has not yet
been described. In this study, eight ascorbate peroxidase (APX) gene families in barley were identified. The
barley APX families were characterized for phylogenetic tree, conserved motifs, gene ontology, correlation of
traits with gene expression, prediction of cis-elements, and gene expression in APX under abiotic stress con-
ditions. In addition, the analysis of enzymes activities was performed on two contrasting Iranian barley culti-
vars namely Sahra (drought tolerant) and Nobahar (drought susceptible) under abiotic stress (PEG, heat,
ABA, and salt) conditions. The Sahra, an early maturing cultivar with a longer awn, has a higher drought
resistance than Nobahar cultivar. Gene expression analysis revealed that 8 HvAPX genes were accumulated in
the leaf and root tissues at 24 and 48 hours after being subjected to abiotic stresses. Furthermore, the gene
expression analysis of the HvAPX genes revealed that gene expression was up/down regulated in response to
PEG-induced drought stress, ABA, salt, and heat stresses in the leaf and root tissues. The phylogenetic anal-
ysis of the HvAPX proteins sequences in barley were grouped into three clusters. The HvAPX7 and HvAPX8
genes had the highest number of cis-elements in their promoter regions, indicating that they might be stimu-
lated by a plethora of environmental stresses. The HvAPX genes had GT1-motif, STRE, CAAT-box, MYB,
and MYC in their promoter regions, playing a key role in response to abiotic stresses. Our findings provide
new insights into APX genes and provide a basis for next investigations of APX genes in plant improvement
(breeding) programs.
Keywords: ascorbate peroxidase, abiotic stresses, barley, catalase, peroxidase
DOI: 10.1134/S1021443723602288
INTRODUCTION
Various environmental stresses lead to excessive
production of ROS causing progressive oxidative
damage and ultimately cell death. The ROS have
comprehensive influence in cell physiology. Plants
contain various enzymatic antioxidants, such as per-
oxidase (POD), catalase (CAT), and ascorbate per-
oxidase (APX). The SOD is also a very important
antioxidant enzyme. In addition, non-enzymatic anti-
oxidants, such as ascorbate (AsA), glutathione, flavo-
noids, and carotenoids are involved in ROS scaveng-
ing system [1]. The APX genes are known to regulate
plant growth processes and response to various stresses
by controlling H2O2 signal transduction, which was
seen in Arabidopsis thaliana cAPX1 mutation with
reduced photosynthesis and delayed flowering as com-
pared to the wild type [2]. In addition, the AtAPX6 pro-
tects A. thaliana seeds against oxidative stress during
drying, maturation, and germination [3]. The effect of
the OsAPX1 gene on seed growth was reported in rice,
where mutant lines showed reduced spike size and
weight and produced 58% aborted seeds. In addition,
the rates of growth and development before flowering
and the number of seeds per spike were similar to the
wild type [4]. The OsAPX2 mutation decreases pleiotro-
pic effects on growth and tolerance to abiotic stress [5].
A high activity of APX was reported in the tolerant
genotype of sweet potato at 24 and 48 hours after
application of salt stress [1]. A previous study showed
that increased expression of AtAPX2 was reported
under high temperature stress [3]. As previously
reported, peroxisomal Populus APX, OsAPX1,
OsAPX2, and OsAPX5-OsAPX7 in Oryza sativa, and
RESEARCH PAPERS
2 Page 2 of 12
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
SAIDI et al.
cytosolic APX2 in Vigna unguiculata showed high
expression during drought stress [1]. Overexpression
of Solanum lycopersicum tAPX increased salt tolerance
and osmotic stress in transgenic tobacco plants [1].
Abiotic stresses such as drought and salinity
increase respiration rate and as a result of electron
leakage and production of ROS including superoxide
radical ( ), hydrogen peroxide (H2O2), and hydroxyl
radical (•OH) lead to higher concentration of these
free radicals, causing toxicity or oxidative damage to
various components of living cells, including fats, pro-
teins and nucleic acids [6]. The activation of the
plant’s antioxidant defense system under oxidative
stress (including drought) has been reported in various
researches [7–9]. Under environmental stress condi-
tions, redox homeostasis in plants is possible through
the antioxidant system. Generally, the detoxification
of free radicals against stresses in plants are classified
into two enzymatic and non-enzymatic systems [9].
The APX is a highly active class of antioxidants and is
a type I carrier peroxidase involved in the ascorbate-
glutathione pathway to remove excess H2O2 in plants
under control and stressed conditions [5]. The number
of APX family genes varies depending on the species.
Based on plant genomic information, the APX family
members have already been reported in several plants
including Arabidopsis, rice, tomato, sorghum and cot-
ton [2–5]. So far, 9 APX genes have been identif ied in
Arabidopsis [10], 8 in O. sativa [2], 7 in L. esculentum
[11], and 26 in G. hirsutum [3].
Barley performance is severely reduced by environ-
mental stresses such as drought, heat, and salinity.
Some researchers proved that heat and drought stress
have severe effects on growth, yield, and quality of
barley [12–14]. Despite the important role of these
genes, systematic characterization of HvAPXs genes in
major barley crop has not been done. In the present
study, we performed a genomic analysis of HvAPX
genes in barley. The identified genes and proteins were
surveyed to a systematic bioinformatics description to
investigate their physicochemical characteristics. The
function of these genes in abiotic stress responses was
analyzed using transcription profiling. Furthermore,
the expression of selected genes among drought tolerant
and sensitive cultivars was confirmed by qRT-PCR. To
confirm their response to abiotic stresses, the APX,
POD, and CAT activity were also measured after
ABA, heat, salt, and PEG-induced drought stresses at
24 and 48 hours after stress application.
MATERIAL AND METHODS
Identification of HvAPX genes in barley. To investi-
gate genome wide analysis, two techniques were used
to identify HvAPX genes in barley. In the first method,
the homology of proteins was identified with APX
proteins from Arabidopsis and rice. The second tech-
nique was to retrieve the APX protein sequence using
2
O
−
i
Hidden Markov Model (HMM) analysis, Pfam num-
ber PF00141 from the Pfam HMM library. The Arabi-
dopsis and rice protein sequences were obtained from
TAIR and RAP-DB databases, respectively. Known
Arabidopsis APX protein sequences were retrieved
from TAIR, and used as search sequences for the
tBLASTn program in barley to search for similar pro-
tein sequences. All putative sequences were checked
against SMART and interproscan databases. The
remaining eight non-redundant candidates were iden-
tified as HvAPX proteins.
Phylogenetic analysis, conserved motifs, subcellular
localization, gene structure analysis of HvAPX genes.
Alignment of protein sequences of Arabidopsis, maize,
Brachypodium distachyon, and barley was performed
using ClustalW method. Phylogenetic tree drawing
was done using MEGA 7.0 software and NJ algorithm.
MEME database was used to identify HvAPX conserved
motifs (http://mee.sdsc.edu/meme/meme.html). The
ExPASy server (http://web.expasy.org/computepi/)
was used to predict the theoretical isoelectric point (pI)
and molecular weight (kDa) of APX proteins. Subcel-
lular localization of HvAPX proteins was performed
using CELLO v.2.5.
Prediction of cis-elements and functional annotation
of HvAPX genes. The PlantCARE database (http://
bioinformatics.psb.ugent.be/webtools/plantcare/html/)
was used to predict the cis-elements and the heatmap
was displayed using TBtools software. Further, 2000 bp
upstream of the promoter region of HvAPX genes was
surveyed.
Plant growth and abiotic-induced expression pro-
files of HvAPX genes. To investigate the expression
patterns of APX genes under abiotic stress, 10 seeds of
two contrasting Iranian barley cultivars were grown in
pots f illed with soil and kept under greenhouse condi-
tions: day/night temperature at 25°C, with 16 hours of
light and 8 hours of dark period was maintained. In
this study, the Sahra and Nobahar cultivars were used
as two contrasting cultivars in response to abiotic
stresses. The Sahra cultivar, a high-yielding drought
tolerant cultivar, is cultivated in most regions of Iran.
In contrast, the Nobahar cultivar is sensitive to
drought. To evaluate the expression of HvAPXs under
abiotic stress, two week-old seedlings were treated
with 20% PEG 6000, salt (200 mM), and heat (42°C)
stresses. Leaves and root from plants were harvested at
24 and 48hours after abiotic stresses and immediately
stored at −80°C for further analysis. The expression
profiles of the eight HvAPX genes were analyzed in two
barley cultivars based on the qRT-PCR data with three
replications. For phyto-hormone treatments, leaves
and root were sprayed with 100μM ABA under control
and stress conditions. Barley cultivars that grew nor-
mally were used as control replicates. We used two
time points of 24 and 48 hours after heat, salt, ABA,
and PEG-induced drought stresses to investigate their
gene expression under these abiotic stresses.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
GENOME-WIDE IDENTIFICATION OF ASCORBATE PEROXIDASE GENE FAMILY Page 3 of 12 2
RNA extraction and quantitative real-time PCR
(qRT-PCR) analysis. Total RNA was extracted from
leaves and roots of barley under control and stress con-
ditions using the RNX-Plus kit according to the man-
ufacturer’s instructions. RNA was extracted from
leaves and roots collected from 2-week-old seedlings
after 24 and 48 hours of abiotic stress treatments. The
purity and concentration of RNA was determined by
NanoDrop and its quality was confirmed using
1% agarose gel analysis. Then cDNA was prepared
according to the instructions of the Easy cDNA Syn-
thesis Kit. For the analysis of each gene, three repeti-
tions were performed, and due to its stable expression
under different environmental stresses, the Actin gene
was utilized as the reference gene. The QRT-PCR was
performed on an ABI 7500 using SYBR Green Super-
mix as described in the manufacturer’s instructions.
Relative expression was determined through the 2–∆∆Ct
technique after normalizing the Ct value for individual
genes against actin [15]. In the current study, the
expression profile for the eight HvAPX genes in leaves
and root under PEG-induced drought stress, ABA,
heat, and salt stresses were determined using qRT-PCR.
Duncan’s test was used to compare the means of treat-
ments at P < 0.05 with three independent biological
replicates. Gene primer sequences for the RT-qPCR
are listed in the Supplementary Table S1.
Characterization of enzymes activity. To investigate
enzymes activity, control and stressed barley leaves
and root were collected at the seedling stage. Root and
leaf tissue samples (0.5 g) were ground into fine pow-
der under liquid nitrogen and then mixed with 10 mL
of pre-cooled phosphate buffer (50 mM, pH 7.8) con-
taining 1% (w/v) PVP. The mixture was centrifuged at
8000 g at 4°C for 40 min. The obtained supernatant
was used for the enzyme assay as cr ude enzyme prepa-
ration. CAT, APX, and POD were measured accord-
ing to Ekinci et al. [16], Nakano and Asada [17], and
Chance and Machly [18], respectively. From the addi-
tion of H2O2, changes in absorbance were monitored
for 120 s at 240, 290, and 470 nm to measure CAT,
APX, and POD activities, respectively.
Statistical analysis. Statistical analysis was per-
formed using SPSS. Differences across tissues were
analyzed using one-way ANOVA test. Duncan’s test
was used to compare the treatment means at P < 0.05.
Values represent the means of three replications per
treatment.
RESULTS AND DISCUSSION
In the present study, bioinformatics study of
HvAPX genes was performed using various tools. The
HvAPX gene names and chemical characteristics are
listed in the Supplementary Table S2. The alignment
results of proteins sequences were performed using
MEGA7 software and the phylogenetic tree was plot-
ted using the NJ algorithm, according to which the
genes were divided into three clusters (Fig. 1). All APX
genes are listed in Supplementary Table S2 along with
gene names, gene details, amino acid length and
amino acid isoelectric point. Bioinformatics analysis
such as phylogenetic relationships and conserved
motifs were performed. According to the intracellular
location of APXs, they can be grouped into cytoplas-
mic APX, peroxisome APX, chloroplast APX, and
chloroplast/mitochondrial APX, however, their local-
ization and are different in cells of different species.
Eight genes of the HvAPX gene family have been rec-
ognized in barley, including two in the cytoplasm
(APX1 and APX2), two in the peroxisome (HvAPX3
and HvAPX4), and three in the chloroplast (HvAPX6,
HvAPX7, and HvAPX8). Our results agreed with other
researchers [19, 20] in that the HvAPX6, HvAPX7 and
HvAPX8 genes play key roles in response to abiotic
stresses. Expression profiles and enzymes activity of
HvAPXs genes were analyzed under abiotic stresses.
The HvAPX genes ranged from 250 to 453 amino acids,
with a predicted isoelectric point (pI) varying from
4.38 to 8.57 with a molecular weight of 24.46 to
48.91 kDa (Supplementary Table S2).
Plants can respond to drought stress with different
morphological, physiological, biochemical or molec-
ular mechanisms. These mechanisms lead to diverse
responses from either enabling plants to become more
tolerant or avoid facing the stress [21]. In some of these
responses, a significant increase in the amount of
reactive oxygen species (ROS) is observed, increasing
the activity of some antioxidant enzymes and antioxi-
dant compounds. Among these antioxidant enzymes,
POD, CAT and glutathione reductase can be men-
tioned [22].
To identify conserved motifs, the APX protein
sequences were determined by MEME database.
Among the APX proteins, three various conserved
motifs were identif ied. The presence of conserved
motifs in the same group probably indicates the related
function of the proteins. In these motifs, the amino
acid length varied from 31 to 50. The number of con-
served domains among the three APX groups is given
in Fig. 2a.
Prediction of Cis-Rlements in HvAPX Genes
To investigate regulatory elements within the pro-
moter region of HvAPX genes associated with abiotic
stresses, the PlantCARE database was utilized to pre-
dict the transcription factor binding sites in the pro-
moter region up to 2000 bp upstream of the ATG of
HvAPX genes. The promoter region analysis indicated
that there are different cis-elements responsive to
light, hormonal, and abiotic stresses. The HvAPX6,
HvAPX7, and HvAPX8 genes had the highest number
of cis-elements in the promoter regions (Fig. 3). Fur-
ther, most of the cis-elements in the upstream region
of the genes are related to cis-elements responding to
abiotic stress. In this study, different cis-elements such
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RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
SAIDI et al.
as GT-1 motif, MYB, MYC, STRE, and light respon-
sive elements (Sp1, A-box, Box4, and box s) were iden-
tified in response to various stresses. Also, cis-elements
responsive to different hormones such as SA-responsive
elements (TCA-element, as-1), Methyl jasmonate ele-
ments (CGTCA-motif and TGACG-motif), ABA-
responsive elements (ABRE), Gibberellin-responsive
element (TGA-element), and Auxin responsive ele-
ments (AuxRR-core) were present in the promoter
region of HvAPX genes. Additionally, some other cis-
elements were expressed in the promoters of HvAPXs
genes including low temperature stress responsive
(Myb, G-box and LTR) and high temperature stress
responsive (WRE3) elements. The Cross-talk among
hormones through cis-elements related to auxin, gib-
berellins (GA), abscisic acid (ABA), and salicylic acid
(S A) pla y cr ucial rol es dur ing plant res pon se to d iff erent
abiotic stresses. With the presence of different cis-ele-
ments in the HvAPX genes, plants adapt to increased
gene expression during its growth in response to vari-
ous environmental stresses [23]. The ABA, a major
phyto-hormone regulating stress responses, interacts
with the JA and SA signaling pathways to respond to
abiotic stresses. The DRE, ABRE, and MYB cis-ele-
ments were identified in the promoters of HvAPX
genes. These signaling factors might be implicated in
the amplification of stress signals in different plant
cells under abiotic stresses. The ABRE and DRE cis-
elements can interact under environmental stresses.
The promoter sequences of HvAPX genes contain sev-
eral cis-elements, such as ABRE, G-box, W-box,
AuXRE, and DRE, which are associated with stress
responses, indicating the potential interaction of abi-
otic stress responsive TFs with cis-elements [23].
According to the study conducted on pepper, most of
the APX genes have cis-elements responsive to light
and abiotic stresses [24].
Gene Ontology of HvAPX Proteins
The gene ontology of HvAPXs showed that the
majority of the HvAPX proteins were involved in oxi-
dative stress, response to stress, cellular process, and
cellular anatomical entity in the biological process.
Membrane and cytoplasm are involved in cellular
Fig. 1. Phylogenetic tree of APX genes created by the neighbor-joining (NJ) method in MEGA7.0 software in Arabidopsis, rice,
maize, and barley.
HvAPX1
HvAPX1b
HvAPX2
HvAPX3
HvAPX4
HvAPX6
HvAPX7
HvAPX8
OsAPX1
OsAPX9
OsAPX2
OsAPX3
OsAPX4
OsAPX6
OsAPX5
OsAPX7
OsAPX8
ZmAPX1
ZmAPX9
ZmAPX2
ZmAPX3
ZmAPX4
ZmAPX5
ZmAPX6
ZmAPX7
ZmAPX8
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
GENOME-WIDE IDENTIFICATION OF ASCORBATE PEROXIDASE GENE FAMILY Page 5 of 12 2
Fig. 2. Conserved APX protein motifs in barley, as recognized by MEME database. Motifs 1–3 are id entified by diff erent colors (a)
and consensus sequence for putative motifs (b).
(b)
0 50 150 250 350100 200 300 400 450 500
HvAPX3
HvAPX2
HvAPX1b
HvAPX1
HvAPX4
HvAPX6
HvAPX7
HvAPX8
(a)
5' 3'
Motif 2
Motif 1
Motif 3
Fig. 3. Cis-element analysis of eight HvAPX genes from the upstream 2000 bp sequence to the transcription start site.
0 200 600 1000 1400400 800 1200 1600 1800 2000
HvAPX3
HvAPX2
HvAPX1b
HvAPX1
HvAPX4
HvAPX6
HvAPX7
HvAPX8
5' 3'
common cis-acting element in promoter and enhancer regions
cis-acting regulatory element involved in the MeJA-responsiveness
cis-acting regulatory element essential for the anaerobic induction
cis-acting element involved in the drought responsiveness
light responsive element
cis-acting element involved in the absasic acid responsiveness
cis-acting regulatory element involved in the SA-responsiveness
cis-acting regulatory element involved in high temprature
cis-acting regulatory element involved in light responsiveness
MYBHvI binding site
cis-acting element involved in low-temperature responsiveness
cis-acting regulatory element involved in auxin responsiveness
cis-acting regulatory element involved in zein metabolism regulation
cis-acting regulatory element related to meristem expression
core promoter element around -30 of transcription start
cis-acting regulatory element involved in the low temprature-responsiveness
WRKY binding site
enhancer-like element involved in anoxic specific inducibility
auxin-responsive element
gibberellin-responsive element
MYB binding site involved in drought-inducibility
cis-regulatory element involved in endosperm expression
cis-acting regulatory element involved in circadian control
2 Page 6 of 12
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
SAIDI et al.
component (Fig. 4). Based on the molecular function,
most of the HvAPXs are involved in POD activity, tet-
rapyrrole binding, and antioxidant activity. Different
kinds of HvAPX are involved in POD activity and
response to stress, implying that the APXs might play
major roles in detoxification of cytoplasm during abi-
otic stresses.
Enzymes Activity in Response to Abiotic Stresses
Based on the analysis of APX activities in the leaf,
it was shown that the APX activity in all treatments
showed a significant increase as compared to the con-
trol in the Sahra cultivar. But, the salt treatment at
48-h did not show significant differences for APX
enzymes activity. In the Nobahar cultivar, the APX
activity showed a significant decrease at 48 hours salt
stress, PEG-induced drought stress at 24 hours, heat
at 24 and 48 hours after applying the stress, but it
showed a significant increase in response to 24 hours of
salt stress treatment. However, it did show significant
differences with the control at 48-h of PEG-induced
drought stress and 24-h of ABA treatments.
Based on the analysis of CAT activity in the leaves,
in the Sahra cultivar, the CAT activity showed a signif-
icant decrease at 48 hours of heat stress, 24 hours of
PEG-induced drought stress, and 24 and 48 hours of
ABA stress. Conversely, the CAT activity showed a
significant increase in response to 24 hours after heat
stress, 24 hours after salt stress and 48 hours after
PEG-induced drought stress. Further, the CAT activ-
ity did not show any significant difference as com-
pared to the control condition at 48-h after salt stress.
In the Nobahar cultivar, the CAT activity showed a
significant decrease at 48 hours after salt stress,
24 hours after PEG-induced drought stress, 48 hours
after ABA stress, and 24 and 48 hours after applying
heat stress. On the other hand, the CAT activity did
not show any significant differences as compared to
the control condition at 24-h after salt and 24 hours
after ABA stresses, as well as, 48 hours after PEG-
induced drought stress.
Based on the analysis of POD activity in the leaf, a
significant increase in the POD activity at 48 hours
after salt treatment and 24 and 48 hours after PEG-
induced drought stress were observed as compared to
the control in the Sahra cultivar. However, it did not
show any significant difference as compared to the
control condition in other treatments. In the Nobahar
cultivar, a significant increase in the POD activity at
24 hours after salt treatment was observed as com-
pared to the control. However, it did not show any sig-
nificant difference as compared to the control condi-
tion in other treatments (Fig. 5).
Based on the analysis of the APX activity in the
root, it was shown that the APX activity in all treat-
Fig. 4. Gene ontology of HvAPX proteins using Blast2GO.
8
6
4
2
0
Enrichment score
Biological process Cellular component Molecular function
biological_process
detoxification
cellular process
metabolic process
response to stimulus
response to chemical
cellular detoxification
catabolic process
cellular response to stimulus
organic substance metabolic process
cellular metabolic process
response to stress
organic substance catabolic process
reactive oxygen species metabolic process
cellular response to chemical stimulus
response to oxidative stress
hydrogen peroxide metabolic process
cellular oxidant detoxification
response to toxic substance
response to oxygen-containing compound
cellular catabolic process
cellular response to stress
cellular response to oxidative stress
cellular response to chemical stress
hydrogen peroxide catabolic process
response to reactive oxygen species
cellular response to toxic substance
cellular_component
cellular anatomical entity
intracellular anatomical structure
membrane
organelle
cytoplasm
intracellular organelle
membrane-bounded organelle
organelle membrane
plastid
microbody
chloroplast
microbody membrane
intracellular membrane-bounded organelle
bounding membrane of organelle
peroxisome
peroxisomal membrane
molecular_functfon
binding
catalytic activity
antioxidant activity
heterocyclic compound binding
organic cyclic compound binding
ion binding
oxidoreductase activity
peroxidase activity
oxidoreductase activity, acting on peroxide as acceptor
L-ascorbate peroxidase activity
cation binding
tetrapyrrole binding
heme binding
metal ion binding
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
GENOME-WIDE IDENTIFICATION OF ASCORBATE PEROXIDASE GENE FAMILY Page 7 of 12 2
ments showed a significant increase as compared to
the control in the Sahra cultivar. In the Nobahar cul-
tivar, the APX activity of all the treatments showed a
significant decrease as compared to the control treat-
ment. Based on the analysis of the CAT activity in the
root, the CAT activity showed a significant increase at
24 and 48 hours after ABA treatment, 24 and 48 hours
after heat treatment, and 24 and 48 hours after PEG-
induced drought stress as compared to the control in
the Sahra cultivar.
No significant differences were observed in the
CAT activity at 24 and 48 hours after salt stress in the
Sahra cultivar. Also, no significant differences were
observed in the CAT activity of all the treatments
except at 24 and 48 hours after PEG-induced drought
stress in the Nobahar cultivar. Based on the analysis of
POD activity in the root, the POD activity at 24 hours
after salt treatment, 24 and 48 hours after heat, and
48 hours after PEG-induced drought stress showed a
significant increase as compared to the control condi-
tion in the Sahra cultivar. No significant differences
were observed for POD activity in other treatments. In
the Nobahar cultivar, a significant increase in POD
activity at 24 and 48 hours after heat treatment and at
48 hours after PEG-induced drought stress were
observed as compared to the control. No significant
differences for POD activity was observed in other
treatments. (Fig. 6).
Plants activate a number of morphological, physi-
ological, and biochemical mechanisms in response to
water stress, among which includes changes in some
enzymes such as peroxidase. The reactive oxygen spe-
cies (ROS) cause membrane damage and lead to a
rapid cellular response to initiate plant defense signal-
ing. The activities of CAT and POD enzymes are
increased during biotic and abiotic stresses to protect
Fig. 5. Activities of APX (a), CAT (b), and POD (c) in the leaf at 24 and 48 h after application of abiotic stresses in the Nobahar (1)
and Sahra (2) cultivars. Values represent the means of three replications per treatment. Different letters demonstrate significant dif-
ferences between treatments (P < 0.05, Duncan’s Multiple Range Test).
0.012
0.008
0.010
0.006
0.004
0.002
0
Treatment
Control
12
(с)
ABA 24h Heat 24h PEG 24h Salt 24h
ABA 48h Heat 48h PEG 48h Salt 48h
POD, Pmol/g fr wt
efg efg efg efg efg efg efg efg efg
fg
ef
de
cd
a
de
b
bc
d
(b)
14
12
8
10
6
4
2
0
Control
12
ABA 24h Heat 24h PEG 24h Salt 24hABA 48h Heat 48h PEG 48h Salt 48h
CAT, Pmol/g fr wt
ef
ef ef
de cd
bcd deghi cijij
efcd ad
ab
(a)
1.2
0.8
1.0
0.6
0.4
0.2
0
Control
12
ABA 24h Heat 24h PEG 24h Salt 24hABA 48h Heat 48h PEG 48h Salt 48h
APX, Pmol/g fr wt
ij
cd aa
cde defg efg efgh efgh efgh
gh gh gh
cde bcd bfg hi
2 Page 8 of 12
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
SAIDI et al.
cells from the potentially dangerous effects of ROS [25].
The APX proteins act as efficient ROS scavengers
against various abiotic stresses. Previous studies
reported that APX genes have regulatory roles with
respect to tolerance to several stresses such as salt,
heat, drought, and oxidative stresses [12, 26, 27]. The
effects of reactive oxygen species (ROS) on the
destruction of nuclear DNA include shape change,
oxidation of oxyribose, and DNA strand breakage and
mutation. Among the various types of ROS compounds,
hydroxyl radicals play more important roles in abiotic
stresses. Oxygen free radicals cause oxidative destruction
of proteins. It has been reported that the destruction
occurs in amino acids structure of proteins [28].
Correlation between Activity and Gene Expression
at 24 and 48 Hours after Different Stresses
The HvAPX4l gene had a positive correlation with
HvAPX8l gene and theHvAPX6l gene had a positive
correlation with HvAPX1r gene. The HvAPX3r gene
had a positive correlation with HvAPX1r and HvAPX8r
Fig. 6. Activates of APX (a), CAT (b), and POD (c) in root at 24 and 48 h after application of abiotic stresses in the Nobahar (1)
and Sahra (2) cultivars. Values represent the means of three replications per treatment. Different letters demonstrate significant
differences between treatments (P < 0.05, Duncan’s Multiple Range Test).
0.012
0.008
0.010
0.006
0.004
0.002
0
Treatment
Control
12
(с)
ABA 24h Heat 24h PEG 24h Salt 24hABA 48h Heat 48h PEG 48h Salt 48h
POD, Pmol/g fr wt
b
cc
c
de de de de de de de de de
cd cd
b
aa
1.2
0.8
1.0
0.6
0.4
0.2
0Control
1
2
(a)
ABA 24h Heat 24h PEG 24h Salt 24h
ABA 48h Heat 48h PEG 48h Salt 48h
APX, Pmol/g fr wt
a
d
dd
bc bbbb
ef de
cd
c
ab ab ab ab ab
14
12
8
10
6
4
2
0Control
1
2
(b)
ABA 24h Heat 24h PEG 24h Salt 24hABA 48h Heat 48h PEG 48h Salt 48h
CAT, Pmol/g fr wt
a
ddd
bb
c
hi hi
ghi
ifgh gh gh
gh e
i
i
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
GENOME-WIDE IDENTIFICATION OF ASCORBATE PEROXIDASE GENE FAMILY Page 9 of 12 2
genes whereas, the HvAPX1r gene had a positive cor-
relation with HvAPX8r gene. The HvAPX8r gene had a
negative correlation with HvAPX1br gene and APXl
had a positive correlation with APXr under stress con-
dition (Fig. 7a). The HvAPX7r gene had a positive cor-
relation with HvAPX1br and HvAPX1l genes had a pos-
itive correlation with HvAPX7r gene. The HvAPX8l
gene had a positive correlation with HvAPX4l gene. On
the other hand, the HvAPX7l gene had a negative cor-
relation with APXl and APXr under control condition
(Fig. 7b).
Gene Expression Analysis of HvAPX Genes
in Barley Cultivars under Abiotic Stresses
In the Sahra and Nobahar cultivars, the expression
profiles of eight HvAPX genes in leaves and root were
determined under abiotic stress conditions using
reverse transcription-PCR (qRT-PCR) quantitative
analysis. Evaluation of genes expression in the Sahra
cultivar in response to abiotic stresses indicated that
most of the genes showed an increased expression
under ABA and PEG-induced drought stresses. To
display the expression of HvAPX in plant leaf and
roots tissues in response to abiotic stresses, their
expression levels were analyzed using heatmap (Sup-
plementary Fig. S1). The data showed that the gene
expression levels of the HvAPX involved in the growth
and development of barley were significantly different
in the leaves and root. Based on the results of gene
expression, the Sahra cultivar showed a higher expres-
sion level of APX genes than the Nobahar cultivar. In
addition, the number of APX genes was higher in the
Sahra cultivar than the Nobahar cultivar. The expres-
sion analysis of the HvAPX genes in the leaves in
response to ABA stress in the Sahra cultivar showed
that HvAPX3 gene showed a higher significant expres-
sion after at 24 hours as compared to than 48 hours
after ABA application (Supplementary Fig. S1). Also,
the HvAPX1 gene showed a significant increase in
expression at 48 hours as compared to 24 hours after
the application of ABA stress. The analysis of the
expression of HvAPX genes in the leaves in response to
heat stress in the Sahra cultivar revealed that the
HvAPX2 expressions were increased after 24 hours as
compared to at 48 hours after stress. Also, the HvAPX3
showed a significant increase in expression 48 hours as
compared at 24 hours after application of heat stress in
the leaves (Supplementary Fig. S1). Also, the HvAPX3
gene showed an increase in expression in the leaves at
48 hours after salt stress and PEG-induced drought
stresses application as compared to 24 hours after stress
application (Supplementary Fig. S1). The HvAPX8
showed a significant increase in expression 24 hours as
compared at 48 hours after application of salt stress in
the leaves. A pervious study showed that most of the
ClAPX genes significantly increased in expression in
watermelon under drought stress [29]. These findings
can help to broaden our perception of APX genes under
different stress conditions, especially abiotic stresses.
In the Sahra cultivar, the expression of HvAPX
(HvAPX1, HvAPX2, HvAPX1b and HvAPX7) genes in
the root showed a significant increase in expression at
48 as compared at 24 hours after applying ABA stress.
Analysis of the expression of HvAPX genes in roots in
Fig. 7. The correlation coeff icients between enzymes activities and gene expression at 24 and 48 hours after different stresses (a) and
under control condition (b). Leaf catalase (CATl), leaf peroxidase (PODl), leaf ascorbate peroxidase (APXl), root catalase (CATr),
root peroxidase (PODr), root ascorbate peroxidase (APXr), leaf HvAPXs (HvAPXl) and root HvAPXs (HvAPXr).
HvAPX81
HvAPX41
САТ1
HvAPX3r
HvAPXlb
HvAPX6r
HvAPX61
HvAPX71
HvAPX2r
HvAPX1r
HvAPX8r
HvAPX31
HvAPXll
HvAPX7r
HvAPX21
HvAPXlbr
APX1
APXr
CATr
HvAPX4r
HvAPX4r
APXr
APX1
HvAPXlbr
HvAPX21
HvAPX7r
HvAPXll
HvAPX31
HvAPX8r
HvAPXlr
HvAPX2r
HvAPX71
HvAPX61
HvAPX6r
HvAPXlb
HvAPX3r
САT1
HvAPX8l
HvAPX41
САТr
(a)
HvAPX81
HvAPX41
САТ1
HvAPX3r
HvAPXlb
HvAPX6r
HvAPX61
HvAPX71
HvAPX2r
HvAPX1r
HvAPX8r
HvAPX31
HvAPXll
HvAPX7r
HvAPX21
HvAPXlbr
APX1
APXr
CATr
HvAPX4r
HvAPX4r
APXr
APX1
HvAPXlbr
HvAPX21
HvAPX7r
HvAPXll
HvAPX31
HvAPX8r
HvAPXlr
HvAPX2r
HvAPX71
HvAPX61
HvAPX6r
HvAPXlb
HvAPX3r
САТr
(b)
1.0
0.5
0
–0.5
САT1
HvAPX8l
HvAPX41
2 Page 10 of 12
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
SAIDI et al.
response to heat stress in the Sahra cultivar revealed
that the HvAPX4 showed a significant increase in
expression 24 hours as compared to 48 hours after
stress application. A significantly higher expression
was observed in the HvAPX1b and HvAPX7 genes at
48 hours after heat application as compared to than
after 24 hours after applying heat stress in root. Exam-
ining the expression of HvAPX genes in the roots of the
Sahra cultivar in response to PEG-induced drought
stress, it was observed that the HvAPX1b and HvAPX7
genes showed an increase in expression at 24 hours as
compared to 48 hours after stress application whereas,
the HvAPX4 gene showed an increase in expression at
48 hours as compared to 24 hours after stress applica-
tion (Supplementary Fig. S1). The expression analysis
of HvAPX genes in the roots of the Sahra cultivar in
response to salt stress showed that the HvAPX2,
HvAPX4 and HvAPX7 genes showed a significant
increase in expression 24 hours after salt stress appli-
cation as compared to 48 hours after applying the
stress (Supplementary Fig. S1). A previous study
showed that the chloroplast APXs are mostly used to
protect the photosynthetic system. Mitochondrial
APXs have a positive role in removing hydrogen per-
oxide produced by fatty acid oxidation [30]. The cyto-
plasmic OsAPX2 gene plays an effective role in main-
taining H2O2 homeostasis [30].
Examining the expression of HvAPX genes in the
leaves of the Nobahar cultivar in response to ABA
stress, the HvAPX2 gene showed increased expression at
48 hours after stress application as compared to 24 hours
after stress application (Supplementary Fig. S2). Exam-
ining the expression of HvAPX genes in the leaves of
Nobahar cultivar in response to heat stress, the
HvAPX1b, HvAPX3, HvAPX4, and HvAPX8 genes had
an increase in expression at 24 as compared to 48 hours
after stress application, whereas, the HvAPX2 gene
showed increased expression at 48 hours as compared
to 24 hours after applying the stress. In the leaves, the
expression of HvAPX3 and HvAPX8 genes showed a
significant increase in expression 48 hours after PEG-
induced drought stress as compared to 24 hours after
PEG-induced drought stress. On the other hand, the
expression of HvAPX2 and HvAPX6 genes showed an
increase in expression 24 hours as compared to
48 hours after salt stress application. Also, the HvAPX8
gene showed an increase in expression in the leaves at
48 hours as compared to 24 hours after salt stress
application (Supplementary Fig. S2).
In the root, the expression of HvAPX4 gene showed
an increase in expression 24 as compared to 48 hours
after ABA stress application. Further, the HvAPX1
gene showed an increase in expression at 48 hours as
compared to 24 hours after applying ABA stress. In
response to PEG-induced drought stress in the roots
of the Nobahar cultivar, the expression of HvAPX3
gene showed a significant increase in expression after
24 hours as compared to 48 hours after stress applica-
tion. In response to salt stress, the expression of
HvAPX3 and HvAPX7 genes showed an increase in
expression 24 hours as compared to 48 hours after
stress application (Supplementary Fig. S2). Examin-
ing the expression of HvAPX genes in the root of the
Nobahar cultivar in response to heat stress, the
HvAPX3, HvAPX6, and HvAPX8 gene showed an
increase in expression at 48 hours after stress applica-
tion as compared to 24 hours after stress application
(Supplementary Fig. S2). Our findings revealed that in
the Sahra cultivar, most of the APX genes showed a
significant increase in expression in response to ABA
stress whereas, in the Nobahar cultivar, most of the
genes showed an increase in expression in response to
heat stress (Supplementary Fig. S2). The CaAPX4
showed higher expression under high temperature,
suggesting that CaAPXs probably played an important
role in the abiotic stress response of pepper, which is
consistent with our findings [24–30].
CONCLUSIONS
The APX is considered to be one of the most wide-
spread antioxidant enzymes in plant cells, and APX
isoforms have a much higher affinity for H2O2 than
CAT, making APXs eff icient scavengers of H2O2 under
stress conditions. Some candidate genes such as
HvAPX3/4/7 and HvAPX8 can be utilized to aid barley
breeding programs designed for developing cultivars
tolerant to abiotic stresses. In our paper, the HvAPX7
and HvAPX8 genes revealed higher expressions under
most of the stress treatments, suggesting that these
genes can be involved in several biological processes.
In the Sahra cultivar, the expression patterns of
HvAPX1, HvAPX3, HvAPX7, and HvAPX1b genes
exhibited very high levels during abiotic stresses. The
expression profiles of HvAPX1, HvAPX2, HvAPX3,
and HvAPX8 genes exhibited high levels during abiotic
stresses and development in the Nobahar cultivar.
These results indicate that the APX genes may play
critical roles in the normal growth of plants and during
abiotic stress conditions.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material
available at https://doi.org/10.1134/S1021443723602288.
ACKNOWLEDGMENTS
The authors are thankful to the Department of Plant
Sciences and Biotechnology, Faculty of Life Sciences and
Biotechnology, Shahid Beheshti University, Tehran, Iran
and Elite National Foundation for providing assistance for
this project work.
CONTRIBUTIONS
A.S., Z.H., and H.G. wrote the main manuscript text
and Z.H. prepared the figures. All authors reviewed the
manuscript.
RUSSIAN JOURNAL OF PLANT PHYSIOLOGY Vol. 71:2 2024
GENOME-WIDE IDENTIFICATION OF ASCORBATE PEROXIDASE GENE FAMILY Page 11 of 12 2
FUNDING
This work was supported by ongoing institutional fund-
ing. No additional grants to carry out or direct this particu-
lar research were obtained.
ETHICS APPROVAL
AND CONSENT TO PARTICIPATE
This work does not contain any studies involving human
and animal subjects.
CONFLICT OF INTEREST
The authors of this work declare that they have no con-
flicts of interest.
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