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Open Access https://doi.org/10.48130/grares-0025-0009
Grass Research 2025, 5: e013
Comparative physiological and transcriptomic analyses of zoysiagrass
(Zoysia japonica) species reveal key metabolic pathways for salt tolerance
Zhenzhen Liu1, Shu Ma1, Xinxin Xu1, Jiayue Sun2, Mingna Li3, Yan Sun1, Kehua Wang1, Peisheng Mao1 and Xiqing Ma1*
1College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China
2Qingdao Haiyuan Turfgrass Co., Ltd., Qingdao 266300, Shandong, China
3Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
* Corresponding author, E-mail: ma2016@cau.edu.cn
Abstract
Soil salinity is a significant environmental challenge that adversely affects plant yield and quality. Zoysiagrass (Zoysia japonica), a member of the Gramineae
family, is highly salt-tolerant, making it an excellent model for studying salt stress response mechanisms. We performed physiological and transcriptomic
analyses on two contrasting Zoysiagrass germplasm accessions under high salt conditions. The salt-tolerant germplasm ST68 demonstrated superior
growth phenotypes, higher chlorophyll and relative water content, greater photochemical efficiency, and lower relative electrolyte leakage and sodium ion
content compared to the salt-sensitive germplasm SS9. Transcriptomic analysis revealed differential expression in pathways involved in photosynthesis,
flavonoid biosynthesis, cell wall macromolecule catabolism, phosphate ion homeostasis, and reactive oxygen species response in the tolerant vs the
sensitive line under salt stress. Notably, the ZjHEMA gene, which encodes glutamyl-tRNA reductase, a rate-limiting enzyme in chlorophyll biosynthesis, was
identified as a key regulator due to its significant upregulation under salt stress in the salt-tolerant germplasm, compared to the sensitive one.
Overexpression of the salt-responsive glutamyl-tRNA reductase gene, associated with chlorophyll metabolism in Zoysiagrass, in Arabidopsis led to increased
salt tolerance, as evidenced by elevated chlorophyll content, relative water content, and photochemical efficiency compared to wild-type plants. Our
findings offer new insights into the mechanisms of salt tolerance in Zoysiagrass, laying a foundation for breeding salt-tolerant germplasm.
Citation: Liu Z, Ma S, Xu X, Sun J, Li M, et al. 2025. Comparative physiological and transcriptomic analyses of zoysiagrass (Zoysia japonica) species reveal key
metabolic pathways for salt tolerance. Grass Research 5: e013 https://doi.org/10.48130/grares-0025-0009
Introduction
Soil salinization, a type of abiotic stress, presents a significant
global challenge, particularly in arid and semi-arid regions where it
drastically curtails agricultural productivity[1−5]. It is estimated that
salt stress and alkalinity impact approximately 1 billion hectares of
land worldwide, constituting 7% of the Earth's surface and over 20%
of all arable land[6,7]. In China alone, soil salinization affects about
4.88% of usable land and 9.91 million hectares of arable land[8]. High
soil salinity impairs plants' ability to absorb water due to osmotic
pressure, disrupts physiological processes by accumulating exces-
sive amounts of specific ions like Na+ and Cl−, and leads to nutrient
imbalances that further reduce plant viability[7,9−11].
Investigating how plants respond to salt stress is crucial for deve-
loping effective management strategies and enhancing their over-
all adaptability[12]. Plants have evolved various morphological, phy-
siological, biochemical, and molecular adaptations to thrive under
salt stress[13]. In terms of physiological mechanisms, the plant alle-
viates salt damage through salt excretion, dilution, accumulation,
and avoidance, and enhances salt tolerance through osmotic regu-
lation and active oxygen scavenging[14]. In the osmotic regulation
mechanism, plant cells maintain water balance by accumulating
organic solutes and inorganic ions. Organic solutes include proline,
betaine, soluble sugars, and polyols, among which proline enhances
hydration by binding to proteins at the hydrophobic end, effec-
tively preventing protein denaturation under osmotic stress. Betaine
plays multiple protective roles by maintaining membrane integrity,
stabilizing enzyme activity, and protecting chloroplast function.
Inorganic ions (K+, Na+, Cl−) protect plant cells by dynamically regu-
lating their osmotic pressure[15−19]. It has been confirmed that the
synergistic effect of solutes significantly improves the stability of
plant cells under salt stress[20]. The antioxidant enzyme system in
plants evolved through long-term evolutionary processes, consti-
tutes a conserved defense mechanism that synergistically com-
plements physiological adaptations to environmental stressors. It
mainly consists of enzymatic antioxidants, such as APX (ascorbate
peroxidase), CAT (catalase), POD (peroxidase), SOD (superoxide dis-
mutase), MDHAR (monodehydroascorbate reductase), DHAR (dehy-
droascorbate reductase), GR (glutathione reductase), GPX (gluta-
thione peroxidase), and GST (glutathione S-transferase), as well as
non-enzymatic antioxidants, including AsA (ascorbic acid), GSH
(glutathione), CAR (carotenoid), α-tocopherol, and some alkaloids
and flavonoids[16]. The activity of antioxidant enzymes in plant
chloroplasts-such as SOD, APX, POD, GPX, GST, and CAT is enhanced
to counteract oxidative damage from excess reactive oxygen spe-
cies under salt stress[12]. At the same time, non-enzymatic antioxi-
dants also play a crucial role in plant tolerance to salt stress. AsA
often acts as a potent antioxidant, directly scavenging reactive oxy-
gen species (ROS) under stress. It not only regulates cellular oxida-
tion but also participates in the formation of alpha-tocopherol[21]. As
an important signaling molecule regulating cellular reduction-
oxidation status, AsA plays a key role in processes such as photosyn-
thesis and mitochondrial electron transfer[21]. Signal transduction
in plants intersects under stress conditions, forming a complex
network that regulates the plant's response to salt stress. Based on
the involvement of Ca2+ in the signaling process, these pathways
can be categorized into two groups: Ca2+-dependent and Ca2+-
independent signal transduction pathways (e.g., MAPK pathway)[16].
Ca2+-dependent signal transduction pathways mainly include
the salt overly sensitive (SOS), abscisic acid (ABA), and
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calcium-dependent protein kinase (CDPK) pathways. In these path-
ways, Ca2+ not only acts as an osmoregulatory molecule but also
functions as a signaling molecule in the salt stress response. It
primarily regulates its concentration in plant cells through Ca2+
channels, triggering a cascade of distinct signaling pathways[22]. As
one of the most important signal transduction pathways in plants,
the MAPK cascade amplifies environmental signals through protein
kinase-catalyzed phosphorylation, thereby transmitting signals from
the cell surface to the interior[23].
Gene function mining is a prerequisite for studying the molecular
mechanisms of plant responses to salt stress. Currently, functional
studies of salt-responsive genes in plants mainly focus on antioxi-
dants, osmotic stress, ion transporters, and signal transduction[16].
It has been found that the salinity tolerance of plants under salt
stress is related to their photosynthetic capacity[24]. For most plants,
salt stress affects chlorophyll structure and reduces chlorophyll
content, leading to a decrease in the photosynthetic rate[25]. This is
mainly achieved by directly affecting the activity and expression
levels of enzymes involved in chlorophyll biosynthesis and
photosynthesis[26]. Under salt stress, the chlorophyll and carotenoid
contents of lentil seedlings were significantly reduced. Exogenous
application of glutamate effectively reduced the accumulation of
Na+ in lentil seedlings, significantly improving seedling survival
rates[27]. Chlorophyll content in plants is significantly correlated with
salt tolerance, and its increase can notably improve salt tolerance[28].
The chlorophyll biosynthesis pathway begins with the conversion
of glutamate to glutamyl-tRNA, with GluTR serving as a key rate-
limiting enzyme in the upstream regulation of 5-aminolevulinic acid
(ALA), playing a crucial role in chlorophyll biosynthesis and plant
growth[29,30]. The mutation of the ZmGluTR1 gene in maize directly
caused the mutant to exhibit leaf yellowing, along with decreased
chlorophyll intermediates and photosynthetic pigments[31]. Cur-
rently, GluTR regulation mainly focuses on transcriptional and post-
translational mechanisms, with its regulatory processes involving
redox regulation, heme, and so on[32−34]. However, the mechanisms
by which GluTR-related genes contribute to plant stress responses
have not yet been reported.
Transcriptome sequencing has identified a large number of diffe-
rentially expressed genes (DEGs) involved in salt stress responses
across various species, including rice (Oryza sativa)[35], maize (Zea
mays)[36], soybean (Glycine max)[37], cotton (Gossypium hirsutum)[20],
barley (Hordeum vulgare)[38], oat (Avena sativa)[39], and black wolf-
berry (Lycium ruthenicum)[40]. The integration of RNA-seq techno-
logy with studies of plant morphology and physiology provides
a comprehensive approach to understanding plant stress
responses[41,42].
Turfgrasses serve crucial economic, environmental, ecological,
recreational, and aesthetic functions. However, salinity poses a sig-
nificant challenge, impairing the growth of both cool- and warm-
season turfgrass species in affected regions[20]. Salt stress markedly
diminishes turfgrass quality, growth, development, and other func-
tional traits by creating a hyperosmotic effect, which reduces the
soil's water potential, making it a significant abiotic factor limiting
turfgrass productivity[43]. Therefore, understanding the stress tole-
rance of turfgrass, especially its salt tolerance, has emerged as a criti-
cal research focus[13]. Zoysia, a member of the Gramineae family, is a
widely utilized warm-season turfgrass genus. Given the conside-
rable variation in salt tolerance among Zoysia species, this genus
serves as an ideal model for studying the mechanisms of salt
tolerance[44−46]. Studies on the salt tolerance of Zoysiagrass mainly
focus on the correlation between salt tolerance identification,
phenotypic traits, physiological responses, the effects of exogenous
substances on salt tolerance, and comparative genomic analyses.
For example, Qian et al.[46] evaluated and identified the salt tole-
rance of 29 Zoysiagrass experimental lines and cultivars, explored
the correlation between leaf texture and salt tolerance and exa-
mined the growth and physiological phenotypes of different Zoysia-
grass germplasm under salt stress[47]. Exogenous application of
calcium and spermidine significantly improved the salt tolerance of
Zoysiagrass, with calcium promoting the expulsion of Na+ ions
through the salt glands[48,49]. Based on comparative genomics, the
genomic characteristics of adaptive evolution in S. dulcis were
studied, revealing that its stress response is linked to genes related
to cytochrome P450 and abscisic acid biosynthesis[50]. However, the
physiological and molecular mechanisms underlying the response
to salt stress in Zoysiagrass germplasm with varying salt tolerance
levels remain less studied and require further exploration. To
address this gap, our study employed physiological assessments
and RNA-Seq analyses to compare salt stress tolerance between two
distinct Zoysiagrass species. Through functional analysis of DEGs, we
investigated the role of the chlorophyll metabolism pathway in
plant responses to salt stress, laying the groundwork for a deeper
understanding of the mechanisms underlying plant responses to
salt stress.
Materials and methods
Plant materials and growth conditions
Following the evaluation of 99 Zoysiagrass accessions[49], the salt-
tolerant germplasm ST68 and the salt-sensitive germplasm SS9 from
Qingdao Haiyuan Turfgrass Co., Ltd. (Qingdao, China) were selected
to study the mechanisms of salt responsiveness. Zoysiagrass germ-
plasm accessions were cultivated in round plastic planters (25 cm in
diameter, 18 cm in height) filled with a 1:1 (v/v) mixture of peat and
vermiculite. They were cultured in an artificial climate chamber with
28/25 °C (day/night) temperatures. The plants were watered weekly
with half-strength Hoagland nutrient solution and trimmed weekly
to maintain a uniform canopy height.
Treatments and experimental design
After 2 months of establishment in the artificial climate chamber,
plants were subjected to salt stress by daily irrigation with increa-
sing concentrations of NaCl in half-strength Hoagland nutrient solu-
tion, starting with 80 mM NaCl for one day, followed by 160 mM
NaCl for another day, and finally 250 mM NaCl to avoid salt shock.
SS9 and ST68 were arranged in a randomized complete block design
with four replicates, and physiological measurements were taken at
0, 3, 6, 9, and 12 d of salt stress (250 mM) or control treatment.
Physiological analysis
Photochemical efficiency (Fv/Fm) was measured using the
method described by Ma et al.[51] with a portable fluorometer (OPTI-
SCIENCES OS-30p+, USA) after a 30 min dark adaptation period.
Relative electrolyte leakage (REL) of the leaves was assessed by
weighing approximately 0.1 g of fresh leaves, which were washed
three times with distilled water and cut into 0.5 cm pieces. These
were placed into a clean centrifuge tube containing 30 mL of dis-
tilled water and shaken at 200 rpm at 28 °C for 24 h. The initial
conductivity (Ci) was measured using an electrical conductivity
meter (METTLER TOLEDO FiveEasy Plus FE38). The samples were
then autoclaved at 121 °C for 30 min and cooled to room tempera-
ture to determine the maximum conductivity (Cmax). The REL of
leaves was calculated as (Ci/Cmax) × 100%[52]. Chlorophyll content
(a + b) was determined following the procedure outlined in a pre-
vious study[53]. Briefly, about 0.1 g of fresh leaves was extracted
in 10 mL of dimethyl sulfoxide (DMSO) in the dark for 72 h. The
chlorophyll content was measured at 663 nm and 645 nm using a
Salt tolerance mechanisms in zoysiagrass species
Page 2 of 12 Liu et al. Grass Research 2025, 5: e013
BECKMAN DU2600 spectrophotometer (Puxi Biological Technology
Co., Beijing, China). Relative water content (RWC) was determined as
described by Ma et al.[54]. The fresh weight (FW), turgid weight (TW),
and dry weight (DW) of the leaf samples were measured indepen-
dently. Approximately 0.1 g of fresh leaves were harvested, imme-
diately weighed, and recorded as the FW. These leaves were then
cut into 0.5 cm pieces, immersed in a centrifuge tube filled with
distilled water, and placed in darkness at 4 °C for 24 h. After this
period, the leaves were removed and weighed to determine the TW.
Subsequently, the soaked leaves were dried at 80 °C for 72 h and
weighed again to obtain the DW. RWC was calculated as (FW −
DW)/(TW − DW) × 100%[55].
Ion content measurements
After exposure to the salt treatment for 9 d, leaf and root Na+ and
K+ ion content were measured separately according to the methods
described by Ma et al.[51]. Fresh leaves and roots (1 g each) were
rinsed with distilled water, dried at 80 °C for 72 h, and ground into a
fine powder. The powdered samples were incinerated in a crucible
preheated to 500 °C for 4 h. After cooling to room temperature, the
ash was dissolved in 10 mL of 0.1 N HCl solution. This solution was
then diluted to 50 mL with distilled water and thoroughly mixed. Ion
analysis was performed using an inductively coupled plasma atomic
emission spectrometer (ICP-AES, 715ES, Varian).
RNA sequencing and transcriptomic analysis
On day 9 of treatment, mature leaves from both the salt-stressed
and control groups of SS9 and ST68 were harvested and imme-
diately ground into a fine powder under liquid nitrogen. Total RNA
was extracted using the TransZol Up Plus RNA Kit, and RNA integrity
was verified on 1% agarose gels to select high-quality samples for
sequencing. Libraries for RNA sequencing were prepared using the
NEB Next Ultra™ RNA Library Prep Kit and sequenced (150-bp
paired-end reads) on an Illumina HiSeq 2500 platform (OE Biotech
Co., Ltd., Shanghai, China). Raw sequencing data were filtered using
Trimmomatic software, and clean reads were aligned to the Zoysia
reference genome available at http://zoysia.kazusa.or.jp/.
Functional annotation of unigenes and analysis of
differentially expressed genes
The filtered high-quality reads were aligned to the reference
genome. HTSeq-count was utilized to calculate the read counts for
each gene, reflecting the baseline expression levels. To account for
variances in gene length and sequencing depth, FPKM (Fragments
Per Kilobase of transcript per Million mapped reads) was applied,
standardizing the expression levels across different genes and
samples to ensure comparability. Differential gene expression analy-
sis was conducted using DESeq, with genes identified as differen-
tially expressed based on a threshold of |Fold Change| ≥ 1.5 and a
p-value < 0.05. The DEGs were subsequently annotated for protein
function using comprehensive databases such as the Kyoto Encyclo-
pedia of Genes and Genomes (KEGG), Gene Ontology (GO), Swiss-
Prot, and the NCBI non-redundant protein (NR) database[56].
Functional validation of the ZjHEMA gene through
gene transformation in Arabidopsis thaliana
Total RNA was extracted from the leaves of Zoysiagrass using an
RNA extraction kit (TransZol Up Plus) and reverse transcription was
performed using a cDNA synthesis SuperMix, which served as a
template for cloning. Specific primers (F: 5'-atggacgagctctacaaggtc
gacATGGCGAGCGCCCCGTCGGC-3' and R: 5'-cggggaaattcgagctcG
GTACCTCAGTTTTGAGTCTTCTCGACCTTGGC-3') were used to amplify
the coding region of the ZjHEMA gene (Zjn_sc00092.1.g01460). The
amplified product was cloned into the pSuper1300 over-expression
vector via SalI and KpnI restriction sites. This expression vector was
constructed through homologous recombination and was intro-
duced into Agrobacterium tumefaciens GV3101 by the freeze-thaw
method. Transformation of Arabidopsis thaliana was carried out
using the floral dip method. Seeds from the T0 generation were steri-
lized and germinated on selective medium (50 μg/mL kanamycin)
following Sun et al.[57] to generate transgenic lines. T3 homozygous
seeds were harvested, and RNA was extracted and analyzed for gene
expression using the 2−ΔΔCᴛ method[57]. The transgenic Arabidopsis
lines (ZjHEMA-4 and ZjHEMA-6) and wild-type (WT) were seeded into
1/2 MS medium and then transferred to small pots after 7 d. One-
month-old seedlings were subjected to an incremental NaCl solu-
tion treatment-starting with 50 mM, followed by 100 mM for one
day each to avoid salt shock, and then maintained at 150 mM for
12 d. Post-treatment, physiological parameters such as Fv/Fm, REL,
chlorophyll content, and RWC were measured as per Ma et al.[54].
Statistical analysis
Data analysis was performed using SPSS 22.0, employing a gene-
ral linear model for variance analysis of measurements including Na+
ion content in leaves and roots, salt gland counts, and other varia-
bles. Significant differences between treatments were determined
using Fisher's least significant difference (LSD) test at p < 0.05 and
p < 0.01, as described by Ma et al.[58]. Data visualization was
conducted using Origin 2019 and TBtools[59].
Results
Morphological changes of Zoysiagrass germplasm
under salt stress
Two contrasting Zoysiagrass lines, the salt-tolerant line ST68 and
the salt-sensitive line SS9-were assessed for morphological and
physiological changes in response to 250 mM NaCl (Fig. 1a−c). After
9 d, notable alterations in leaf phenotype were observed in SS9
under salt stress (SS9 + NaCl) compared to the untreated SS9
control, including foliar tip burning, wilting, curling, and water loss.
In contrast, ST68 exhibited no obvious changes in leaf phenotype
under salt stress compared to the untreated control (Fig. 1b). By day
12, SS9 showed stunted growth, pronounced leaf wilting, drying,
and eventually plant death under salt treatment, whereas ST68
exhibited only slight leaf dehydration and drooping under the same
conditions (Fig. 1c).
Physiological responses of Zoysiagrass germplasm
under salt stress: chlorophyll, RWC, photochemical
efficiency, REL, and sodium ion content
Physiological performance including chlorophyll content, RWC,
REL, photochemical efficiency (Fv/Fm), and leaf and root Na+ ion
content, were recorded on days 0, 3, 6, 9, and 12 following the salt
treatment (Fig. 2). The results indicate that the resistant line ST68
exhibited overall better performance under salt stress compared to
the salt-sensitive line SS9. For example, chlorophyll (a + b) content
in SS9 was 71.74% lower on day 9 and 69.89% lower on day 12
compared to the non-stressed control. In contrast, ST68 showed no
significant difference on day 9, but had a 47.55% increase on day 12
compared with its untreated control (Fig. 2a). The RWC of SS9
decreased by 15.65% and 21.58% on days 9 and 12, respectively,
under salt stress compared with the non-stress control, while that of
ST68 remained similar to the untreated control throughout the
experiment (Fig. 2b). The Fv/Fm ratio in SS9 was reduced by 6.42%
and 18.52% compared to the control on days 9 and 12, respectively,
whereas that of ST68 showed no significant difference between the
treated and untreated groups (Fig. 2c). Moreover, salt stress led to
an increase in REL in SS9 (1.96-fold on day 9 and 2.43-fold on day 12
compared to untreated controls) than in ST68 (1.35-fold on day 9
Salt tolerance mechanisms in zoysiagrass species
Liu et al. Grass Research 2025, 5: e013 Page 3 of 12
ST68 SS9 ST68 + NaCl SS9 + NaCl
c
b
a
ST68 SS9 ST68 + NaCl SS9 + NaClST68 SS9 ST68 + NaCl SS9 + NaCl
Fig. 1 Phenotypic change of ST68 and SS9 under salt treatment. (a), (b), and (c) show plants treated with 250 mM NaCl for 0, 9, and 12 d, respectively.
0
5
10
15
20
25
30
35
40 ST68
SS9
ST68 + NaCl
SS9 + NaCl
e
Na+ content (mg·g−1 DW)
Leaf
0
2
4
6
8
10
12
f
Na+ content (mg·g−1 DW)
Root
0 3 6 9 12
−1
0
1
2
3
4
ST68
SS9
ST68 + NaCl
SS9 + NaCl
a
Chlorophyll (a + b) content
(mg·g−1 DW)
0 3 6 9 12
60
100
95
90
85
80
75
70
65
b
Relative water content (%)
036
Days of treatment
9 12
0.8
0.6
0.4
0.2
c
Photochemical efficiency
(Fv/Fm)
0 3 6 9 12
10
80
70
60
50
40
30
20
d
Relative electrolyte leakage (%)
Days of treatment
Fig. 2 Physiological performances of ST68 and SS9 under salt stress (250 mM NaCl). (a) Changes in chlorophyll content under salt stress. (b) Changes in
relative water content (RWC) under salt stress. (c) Changes in photochemical efficiency (Fv/Fm) under salt stress. (d) Changes in relative electrolyte leakage
(REL) under salt stress. (e) Sodium ion content in leaves after 9 d of salt stress. (f) Sodium ion content in roots after 9 d of salt stress. Values of (a)−(d) are
means ± LSD and values of (e), (f) are means ± SD, all from four replicates. Different lowercase letters indicate significance at p < 0.05.
Salt tolerance mechanisms in zoysiagrass species
Page 4 of 12 Liu et al. Grass Research 2025, 5: e013
and ~1.6 fold on day 12 compared to untreated controls) (Fig. 2d).
Furthermore, by day 9 of salt treatment, Na+ ion content signifi-
cantly increased by 4.89- and 7.73-fold in the leaves of ST68 and SS9
compared with untreated controls; notably, the Na+ content was
significantly higher by 23% in SS9 compared to ST68 under stress,
suggesting that fewer Na+ ions accumulate in the leaves of ST68,
thus helping to maintain cellular homeostasis under salt stress
(Fig. 2e). Similarly, Na+ content in the roots of ST68 and SS9 increa-
sed by 8.5- and 8.89-fold, respectively, compared with untreated
controls, however, no significant differences were observed
between the two lines under either treated or untreated conditions
(Fig. 2f).
Scanning electron microscope observation of ST68
and SS9 under salt stress
The distribution of salt glands on the leaf surfaces of ST68 and SS9
was examined using scanning electron microscopy (SEM). Salt
glands were observed on the leaves of both ST68 and SS9 under salt
stress, however, significant differences in gland density were noted
(Fig. 3a−c). Specifically, the leaves of ST68 exhibited 1.89-fold more
salt glands compared to those of SS9 (Fig. 3a & b). Despite these
significant differences in the density of salt glands between the two
lines, no significant differences were detected between the salt-
stressed plants and their respective control plants within each line
(Fig. 3c).
Analysis of DEGs between SS9 and ST68 under salt
stress
To elucidate the molecular mechanisms underlying the salt tole-
rance of the two contrasting Zoysiagrass germplasm accessions,
mature blades of ST68 and SS9 under different conditions were
collected, mixed, and subjected to transcriptome sequencing. The
analysis yielded a total of 96.43 Gb of clean reads with a Q30
percentage of 94.77% and an average GC content of 54.43%. The
RNA data from the three biological replicates demonstrated a high
correlation in gene expression levels (R2 > 0.98), confirming the relia-
bility of the dataset. These reads were then assembled and used for
subsequent analyses (Supplementary Table S1).
As shown in Fig. 4a, a total of 5,243 DEGs were identified in
SS9 under salt stress, with 2,275 up-regulated and 2,968 down-
regulated, compared to its non-stressed control. Similarly, ST68 exhi-
bited 7,315 DEGs, including 3,854 up-regulated and 3,461 down-
regulated genes, under the same conditions. Additionally, under
untreated control conditions, 5,647 DEGs (2,727 up-regulated and
2,930 down-regulated) were observed between SS9 and ST68,
compared to 10,141 DEGs (4,649 up-regulated and 5,492 down-
regulated) between the two lines under salt stress (Fig. 4a). A Venn
diagram was utilized to illustrate the similarities and differences in
gene expression between the germplasm accessions (Fig. 4b). These
findings highlight that the DEGs are germplasm-specific, with ST68
displaying a greater number of DEGs than SS9 when subjected to
salt stress.
GO analysis of DEGs
To further investigate differential gene expression between ST68
and SS9 in response to salt stress, DEGs were functionally catego-
rized by GO enrichment analysis. The analyses of DEGs from SS9 +
NaCl vs SS9, ST68 + NaCl vs ST68, SS9 vs ST68, and SS9 + NaCl vs
ST68 + NaCl comparisons revealed enrichments in 293, 285, 203,
and 217 GO terms, respectively (Supplementary Table S2). This anal-
ysis reveals germplasm-specific enrichment patterns, highlighting
differences in biological processes (BP), cellular components (CC),
and molecular functions (MF) between SS9 and ST68 under salt
stress conditions. Compared to the susceptible line SS9 (3,330
DEGs), ST68 exhibited a higher number of DEGs (4,366) before and
after salt stress, representing a 1.31-fold increase. The most signifi-
cant GO terms associated with DEGs between the untreated and
treated groups of SS9 included nitrate assimilation (GO:0042128),
chloroplast (GO:0009507), and nitrate transmembrane transporter
activity (GO:0015112) (Supplementary Fig. S1a). In contrast, the top
GO assignments for the ST68 vs ST68 + NaCl comparison included
chitin (GO:0010200), an integral component of membrane (GO:
0016021), and sequence-specific DNA binding (GO:0043565)
(Supplementary Fig. S1b).
Response to heat (GO:0009408), chloroplast (GO:0009507), and
beta-amylase activity (GO:0016161) were the top pathways up-
regulated in both SS9 + NaCl and ST68 + NaCl compared to their
0
20
10
30
40
50
60
ST68 SS9 ST68 + NaCl SS9 + NaCl
c
b
400 μm
a
400 μm
Density of salt gland (number mm−2)
Fig. 3 Distribution and quantification of salt glands in two Zoysiagrass
germplasm accessions (ST68 and SS9) under salt stress. (a) Distribution
of salt glands on the leaves surface in ST68 + NaCl. (b) Distribution of
salt glands on the leaves surface in SS9 + NaCl. (c) Density of salt glands
in ST68 and SS9 with and without NaCl treatment. The values are means
± SD from six replicates. Different lowercase letters indicate significance
at p < 0.05.
Salt tolerance mechanisms in zoysiagrass species
Liu et al. Grass Research 2025, 5: e013 Page 5 of 12
respective controls (Table 1). Common down-regulated pathways
between SS9 + NaCl and ST68 + NaCl compared to their respective
controls included chitin response (GO:0010200), an integral compo-
nent of membrane and protein serine or threonine kinase activity
(Table 1). Uniquely up-regulated DEGs in the group SS9 + NaCl vs
ST68 + NaCl, compared with the group SS9 vs ST68, were observed
in photosynthesis (GO:0015979), chloroplast thylakoid membrane
(GO: 0009535), and chlorophyll binding (GO:0016168) (Table 2). Con-
versely, uniquely down-regulated DEGs under the same conditions
included L-phenylalanine, integral component of membrane, and
phenylalanine ammonia-lyase activity. This analysis highlights that
SS9 and ST68 display unique gene expression profiles and pathway
activations under salt stress, revealing key molecular mechanisms
that differentiate their responses.
KEGG enrichment analysis of DEGs
To further explore the response mechanisms of the two Zoysia
germplasm accessions under salt stress, we analyzed the KEGG
enrichment pathways of DEGs between the two germplasm acces-
sions under salt stress and control conditions. The KEGG enrich-
ment analysis revealed that DEGs between the two Zoysiagrass
germplasm accessions under salt stress were significantly enriched
in pathways associated with photosynthesis, redox homeostasis,
and carbohydrate metabolism (Table 3). Notably, the most highly
enriched pathway was photosynthesis (ko00195), with 42 DEGs
involved, suggesting that salt tolerance may correlate with differen-
tial regulation of photosynthetic electron transport and chlorophyll
biosynthesis. This is further supported by the enrichment of photo-
synthesis-antenna proteins (ko00196) and porphyrin and chloro-
phyll metabolism (ko00860). Additionally, pathways linked to anti-
oxidant defense and osmotic adjustment, such as glutathione
metabolism (ko00480), starch and sucrose metabolism (ko00500),
amino sugar and nucleotide sugar metabolism (ko00520) were
significantly enriched, implying coordinated regulation of ROS
scavenging and osmolyte synthesis under salt stress. Intriguingly,
nicotinate and nicotinamide metabolism (ko00760) also exhibited
strong enrichment, potentially reflecting energy metabolism adap-
tation to salinity.
DEGs between SS9 and ST68 are enriched for
chlorophyll metabolism-related genes under salt
stress
The notable upregulation of the chlorophyll metabolism path-
way under salt stress prompted further investigation. Differential
expression analysis revealed that the SS9 + NaCl vs SS9 comparison
exhibited a greater number of DEGs associated with chlorophyll
a
b
SS9 + NaCl vs ST68 + NaC1
SS9 + NaCl vs SS9
ST68 + NaCl vs ST68
SS9 vs ST68
Down-regulated
Up-regulated
−6,000
−4,000
−2,000
Number of DEGs
2,000
4,000
6,000
0
SS9 + NaCl vs ST68 + NaC1SS9 + NaCl vs SS9
ST68 + NaCl vs ST68 SS9 vs ST68
2,487
2,924
1,718
1,188 256
559
622
518
990
342
992
601
642
663
1,609
Fig. 4 DEGs identified in ST68 and SS9. (a) Number of DEGs in ST68 and SS9 under unstressed and stressed conditions. (b) Venn diagram illustrating the
overlap and unique DEGs between ST68 and SS9 under salt stress.
Table 1. GO terms enriched for DEGs common to SS9 and ST68 in response to salt stress.
Category Term ST68 + NaCl vs ST68 SS9 + NaCl vs SS9
Name ID Pop hits p-adjust List hits List total p-adjust List hits List total
Commonly up-regulated DEGs
BP Response to heat GO:0009408 177 1.66E-07 40 2374 2.34E-03 19 1,352
CC Chloroplast GO:0009507 1,999 5.92E-09 273 4.02E-51 283
MF Beta-amylase activity GO:0016161 18 1.40E-07 10 1.62E-02 3
Commonly down-regulated DEGs
BP Response to chitin GO:0010200 170 1.08E-18 55 1,992 2.25E-05 33 1,978
CC Integral component of membrane GO:0016021 4,892 1.49E-13 542 5.15E-22 579
MF Serine/threonine kinase activity GO:0004674 611 7.06E-07 90 1.65E-03 76
Table 2. GO terms enriched for DEGs unique to each Zoysiagrass line in response to salt stress.
Category GO term ID Pop hits Adjusted p-value List hits List total
Uniquely up-regulated DEGs
BP Photosynthesis GO:0015979 112 1.44E-15 45 2,568
CC Chloroplast thylakoid GO:0009535 400 2.06E-12 93
MF Chlorophyll binding GO:0016168 35 3.79E-07 16
Uniquely down-regulated DEGs
BP L-phenylalanine catabolic process GO:0006559 24 3.50E-08 15 3,143
CC Integral component of membrane GO:0016021 4,892 1.31E-08 769
MF Phenylalanine ammonia-lyase activity GO:0045548 18 3.95E-06 11
Salt tolerance mechanisms in zoysiagrass species
Page 6 of 12 Liu et al. Grass Research 2025, 5: e013
metabolism compared to ST68 + NaCl vs ST68 (Fig. 5). Specifically, a
total of 20 chlorophyll metabolism-related DEGs (18 up-regulated
and two down-regulated genes) were identified in SS9 + NaCl, and
seven such DEGs (four up-regulated and three down-regulated
genes) in ST68 were enriched under salt stress conditions compared
to their respective controls. Additionally, 14 chlorophyll metabolism-
related DEGs (seven up-regulated and seven down-regulated genes)
under normal conditions and 23 chlorophyll metabolism-related
DEGs (19 up-regulated and four down-regulated genes) under salt-
stress conditions were identified in SS9 compared to ST68 (Fig. 5).
Under salt stress conditions, genes encoding glutamyl-tRNA
reductase, magnesium chelatase, and protochlorophyllide oxidore-
ductase exhibited significant differential expression in SS9 and
ST68 compared to their respective controls. Comparative analysis
between SS9 and ST68 under the salt stress condition revealed
that genes encoding key enzymes in chlorophyll metabolism,
such as glutamyl-tRNA synthetase, glutamate-1-semiadehyde 2,1-
aminomutase, uroporphyrinogen III synthase, uroporphyrinogen III
decarboxylase, coproporphyrinogen III oxidase, chlorophyll syn-
thase, and chlorophyll b reductase were specifically expressed in
SS9 compared to ST68 under salt stress conditions. Among these
DEGs, the expression levels of ZjHEMD, ZjHEMF, ZjCHLM, ZjPOR, and
ZjCLH in SS9 were more than 2-fold higher than in ST68, suggesting
these genes may play a crucial role in enhancing salt tolerance.
Notably, two genes encoding glutamyl-tRNA-reductase (ZjHEMA)
and involved in chlorophyll metabolism were differentially expres-
sed between the two lines under salt stress. Specifically, upon salt
treatment, the expression level of Zjn_sc00086.1.g00930 remained
unchanged in ST68 but increased by 1.55-fold in SS9, whereas that
of Zjn_sc00092.1.g01460 decreased in both lines, with a greater
reduction of 4.11-fold observed in SS9 compared to ST68. These
findings reveal that SS9 demonstrates a heightened activation of
chlorophyll metabolism-related genes than ST68 under salt stress.
Overexpression of ZjHEMA enhances salt tolerance in
transgenic Arabidopsis
Glutamyl-tRNA reductase is a key rate-limiting enzyme in the
synthesis of tetrapyrrole during chlorophyll biosynthesis, and its
gene expression differs between ST68 and SS9 under salt treatment.
We verified the function of one ZjHEMA gene (Zjn_sc00092.1.
g01460) in regulating salt stress responses through gene transfor-
mation in Arabidopsis. Seven independent Arabidopsis transgenic
lines were obtained, and two lines that showed the highest ZjHEMA
expression levels were selected for subsequent analyses (Fig. 6a).
Growth and morphological and physiological parameters of ZjHEMA
over-expression (OE) lines were assessed 12 d after treatment with
150 mM NaCl (Fig. 6b).
Under salt stress, the decrease in chlorophyll (a + b) content was
significantly lower in the ZjHEMA OE lines compared to WT. Specifi-
cally, chlorophyll content in ZjHEMA-4 and ZjHEMA-6 decreased by
only 16.73% and 16.67%, respectively, in contrast to a substantial
54.75% reduction in WT, with ZjHEMA-4 and ZjHEMA-6 maintaining
2.08-fold and 1.93-fold higher chlorophyll content than WT, respec-
tively (Fig. 6c). Similarly, the reduction in leaf RWC under salt stress
was much less pronounced in the OE lines than in WT-the RWC
decreased by only 5.84% and 5.89% in ZjHEMA-4 and ZjHEMA-6,
respectively, compared to a 17.55% decrease in WT. As a result, RWC
in the OE lines was 7.37% higher than in WT, suggesting improved
water retention capability of the OE lines under salt stress (Fig. 6d).
While the WT exhibited a significant 21.88% reduction in Fv/Fm
under salt stress, the OE lines showed no discernible difference
between salt stress and control conditions, and the Fv/Fm ratios of
ZjHEMA-4 and ZjHEMA-6 were 1.29-fold higher than the WT under
stress conditions (Fig. 6e). Moreover, increased REL was observed for
all lines under salt stress compared to control conditions; however,
the increased in REL was much less prominent for the OE lines than
WT. Specifically, the REL of ZjHEMA-4 and ZjHEMA-6 was 52.02% and
45.30% lower than that of the WT (Fig. 6f), suggesting better
membrane stability and cellular integrity under salt stress condi-
tions. These results confirm that overexpression of ZjHEMA signifi-
cantly enhances salt stress tolerance in Arabidopsis, supporting its
critical role in salt tolerance.
Discussion
Salt stress represents a significant environmental constraint that
can disrupt osmotic potential and ion levels in plants, ultimately
inhibiting cellular metabolism[60]. In high-salt environments, plants
have evolved various mechanisms to combat salt stress. These
include compartmentalizing ions into salt bladders, secreting excess
Na+ ions through salt glands, or blocking ion transport to above-
ground parts via the rhizome junction, thereby reducing Na+ ion
toxicity[61,62]. This study demonstrated that the salt-tolerant Zoysia-
grass variety ST68 exhibited superior growth phenotypes, higher
chlorophyll content, increased RWC, enhanced photochemical effi-
ciency, reduced REL, and lower Na+ ion content compared to the
salt-sensitive Zoysiagrass germplasm SS9. Further gene expression
analysis revealed that overexpression of a gene associated with salt
response and photosynthesis in Arabidopsis resulted in enhanced
salt tolerance.
Our results indicate that the salt-tolerant Zoysiagrass line ST68
has a higher density of salt glands and lower levels of Na+ ions in the
leaves compared to the salt-sensitive line SS9. This finding aligns
with a previous report that salt resistance in Zoysiagrass is positively
correlated with the density of salt glands and the rate of salt
secretion[63]. The lower Na+ content and higher density of salt
glands may suggest that the tolerant Zoysiagrass line ST68 effec-
tively secretes excess Na+ ions from the cells through salt glands,
thereby maintaining stability in intracellular ion concentrations.
Conversely, with fewer salt glands, the salt-sensitive line SS9
accumulates excess Na+ ions, leading to symptoms such as leaf tip
Table 3. KEGG enrichment analysis for DEGs of two Zoysiagrass germplasm
accessions under salt stress.
Pathway
ID Pathway term Associated
genes
number
Enrichment
score Adjusted
p-value
ko00195 Photosynthesis 42 2.490597262 8.70494E-10
ko00760 Nicotinate and
nicotinamide
metabolism
16 2.424708445 0.000766994
ko00680 Methane metabolism 39 1.67974868 0.002913442
ko00860 Porphyrin and
chlorophyll
metabolism
30 1.753583786 0.00503669
ko00196 Photosynthesis-
antenna proteins 15 2.116394225 0.00671243
ko00520 Amino sugar and
nucleotide sugar
metabolism
76 1.394479184 0.00671243
ko00500 Starch and sucrose
metabolism 109 1.307902668 0.008129324
ko00480 Glutathione
metabolism 53 1.417384716 0.021540316
ko00471 D-Glutamine and D-
glutamate metabolism 3 3.068771626 0.046258729
ko00514 Other types of O-
glycan biosynthesis 4 2.727797001 0.048753999
ko00902 Monoterpenoid
biosynthesis 9 2.045847751 0.048753999
Salt tolerance mechanisms in zoysiagrass species
Liu et al. Grass Research 2025, 5: e013 Page 7 of 12
burning, curling with water loss, and wilting. This mechanism is
supported by similar findings in Rhodes grass, where the leaf blades
secrete large amounts of salt crystals under salt stress, effectively
alleviating salt-induced leaf senescence and physiological
damage[64,65].
The observation that ST68 exhibited greater RWC, Fv/Fm, and
chlorophyll (a + b) content compared to SS9 aligns with findings in
salt-tolerant Bromus inermis under similar conditions[66].
Transcriptome analysis revealed enrichment of DEGs in pathways
related to chloroplasts, beta-amylase activity, serine/threonine
kinase activity, and response to chitin in both salt-sensitive and salt-
tolerant plants under salt stress conditions. Among these, genes
involved in the serine/threonine kinase activity metabolic pathway
are significantly associated with plant salt tolerance[67]. However,
the two Zoysia germplasm accessions displayed distinct response
mechanisms, with ST68 showing upregulation of genes related to
9 + NaCl vs 9
9 vs 68
9 + NaCl vs 68 + NaCl
68 + NaCl vs 68
9 + NaCl vs 9
9 vs 68
9 + NaCl vs 68 + NaCl
68 + NaCl vs 68
−6
−4
−2
0
2
4
6
Log1.5(FC)
Zjn_sc00098.1.g00540
Zjn_sc00016.1.g00560
Zjn_sc00026.1.g00600
Zjn_sc00015.1.g08340
Zjn_sc00108.1.g00190
Zjn_sc00697.1.g00010
Zjn_sc00182.1.g00130
Zjn_sc00098.1.g00540
Zjn_sc00016.1.g00560
Zjn_sc00002.1.g07770
Zjn_sc00004.1.g13610
Zjn_sc00057.1.g01310
Zjn_sc00013.1.g08030
Zjn_sc00048.1.g00940
Zjn_sc00004.1.g09820
Zjn_sc00023.1.g02830
Zjn_sc00112.1.g01010
Zjn_sc00133.1.g00260
Zjn_sc00002.1.g02040
Zjn_sc00036.1.g01950
Zjn_sc00003.1.g01070
Zjn_sc00002.1.g12360
Zjn_sc00003.1.g10240
Zjn_sc00003.1.g10250
Zjn_sc00086.1.g00930
Zjn_sc00152.1.g00710
Zjn_sc00043.1.g02690
Zjn_sc00092.1.g01460
Zjn_sc00012.1.g07860
Zjn_sc00003.1.g01080
Zjn_sc00043.1.g03030
Zjn_sc00200.1.g00030
Zjn_sc00003.1.g00580
Fig. 5 Expression profiles of DEGs related to chlorophyll metabolism. EARS, glutamyl-tRNA synthetase; HEMA, glutamyl-tRNA reductase; HEML,
glutamate-1-semialdehyde 2,1-aminomutase; HEMB, porphobilinogen synthase; HEMC, hydroxymethylbilane synthase; HEMD, uroporphyrinogen III
synthase; HEME, uroporphyrinogen III decarboxylase, HEMF, coproporphyrinogen III oxidase; HEMG, protoporphyrinogen oxidase; CHLD, magnesium
chelatase; CHLM, magnesium proto IX methyltransferase; CRD1, Mg-protoporphyrin IX monomethyl ester cyclase; POR, protochlorophyllide
oxidoreductase; DVR, 3,8-divinyl protochlorophylide a 8-vinyl reductase; CAO, chlorophyllide a oxygenase; CHLG, chlorophyll synthase; NYC1, chlorophyll
b reductase; HCAR, 7-hydroxymethyl chlorophyll a reductase.
Salt tolerance mechanisms in zoysiagrass species
Page 8 of 12 Liu et al. Grass Research 2025, 5: e013
photosynthesis, chloroplast thylakoids, and chlorophyll binding.
This adaptation likely facilitates enhanced CO2 concentration and
chloroplast function under salt stress, critical for maintaining photo-
synthesis and mitigating oxidative stress by eliminating excessive
reactive oxygen species generated under such conditions[68−70].
Based on the transcriptome sequencing of ST68 and SS9 under salt
stress, we discovered that DEGs in salt-tolerant germplasm ST68 and
salt-sensitive germplasm SS9 were enriched in three photosynthetic
pathways, such as photosynthesis (ko00195), porphyrin and chloro-
phyll metabolism (ko00860), and photosystem-antenna proteins
(ko00196). This finding is consistent with research by Ryu & Cho,
which suggests that salt tolerance in plants increases with enhanced
c d
Chlorophyll (a + b) content (mg·g−1 DW)
Relative water content (%)
e f
Relative electrolyte leakage (%)
Photochemical efficiency
ab
Relative expression level
Fig. 6 Morphological and physiological responses of ZjHEMA overexpression Arabidopsis lines under salt stress. (a) Relative expression levels of ZjHEMA
in Arabidopsis transgenic lines. AtUBQ served as the endogenous control. (b) Plant morphologies of the WT and ZjHEMA OE lines under salt stress.
(c) Chlorophyll (a + b) contents in the WT and ZjHEMA OE lines. (d) RWC of the WT and ZjHEMA OE lines. (e) Fv/Fm ratios of WT and ZjHEMA OE lines. (f) REL
of the WT and ZjHEMA OE lines. The values are means ± SD from four replicates. Different uppercase letters indicate significance at p < 0.01.
Salt tolerance mechanisms in zoysiagrass species
Liu et al. Grass Research 2025, 5: e013 Page 9 of 12
photosynthesis[24]. Zhao et al. found that overexpression of the
light-harvesting chlorophyll a/b-binding protein MdLhcb4.3 in
apple significantly enhanced the tolerance of transgenic apple
callus and transgenic Arabidopsis to osmotic stress[71]. Additionally,
DEGs in salt-tolerant germplasm ST68 and salt-sensitive germplasm
SS9 were significantly enriched in the glutathione metabolic path-
way (ko00480), where NADPH-dependent glutathione reductase
catalyzes the reduction of glutathione, removes excessive ROS gene-
rated under salt stress, and enhances plant salt tolerance[72].
Notably, we validated a key gene in the chlorophyll metabolic
pathway that was differentially expressed between SS9 and ST68
under salt stress conditions. Sequence analysis identified this gene
as ZjHEMA1, one of three HEMA genes encoding glutamyl-tRNA
reductase, a rate-limiting enzyme essential for the synthesis of tetra-
pyrrole compounds, such as chlorophyll, predominantly expressed
in the green parts of plants. Previous studies have revealed that
GluTR exists in multiple copies in higher plants, and it is encoded by
three HEMA genes (HEMA1, HEMA2, HEMA3) in Arabidopsis thaliana,
with the GluTR1 and GluTR2 subtypes being predominant. Among
them, GluTR1 is encoded by HEMA1 and is primarily expressed in the
green parts of plants[31,33]. Overexpression of ZjHEMA1 in Arabidop-
sis resulted in enhanced salt tolerance compared to the WT, mirro-
ring the effects seen in OsHEMA1 overexpression lines in rice under
salt stress[68]. The salt tolerance associated with ZjHEMA1 seen here
is supported by the role of glutamyl-tRNA reductase in ALA pro-
duction. Glutamyl-tRNA reductase catalyzes the conversion of L-
glutamyl-tRNA to L-glutamic acid-1-semialdehyde (GSA), releasing
tRNA-a key regulatory step in chlorophyll synthesis[73]. The unstable
intermediate GSA is subsequently isomerized to ALA by GSA
aminotransferase[68,74]. ALA serves as the precursor for all tetra-
pyrroles and protochlorophyllide, which converts to chlorophyll
upon exposure to light[75]. ALA is also known to enhance plant stress
tolerance[76,77]. However, the specific mechanisms by which ALA
enhances salt tolerance in plants warrant further investigation.
Conclusions
Soil salinization is a serious global problem that can severely limit
agricultural production. In this study, we analyzed two contrasting
Zoysiagrass germplasm accessions, the salt-tolerant ST68, and the
salt-sensitive SS9, under high salt conditions. ST68 exhibited supe-
rior growth, higher chlorophyll, and water content, and enhanced
photochemical efficiency. Additionally, transcriptomic analysis revea-
led differential expression in key pathways such as photosynthesis
and flavonoid biosynthesis. Moreover, we validated the role of one
DEG between ST68 and SS9, a salt-responsive glutamyl-tRNA reduc-
tase gene involved in chlorophyll metabolism, in conferring salt
tolerance through Arabidopsis transformation experiments. The
findings of this research provide valuable insights into the physio-
logical and molecular mechanisms underlying salt tolerance in
Zoysiagrass and a candidate gene for improving salt resistance
in Zoysiagrass and related species through breeding or genetic
engineering.
Author contributions
The authors confirm their contribution to the paper as follows:
study conception and design: Ma X; data collection: Liu Z, Ma S, Xu
X, Sun J; analysis and interpretation of results: Liu Z, Ma X, Sun Y;
draft manuscript preparation: Liu Z, Li M, Mao P, Wang K, Ma X. All
authors reviewed the results and approved the final version of the
manuscript.
Data availability
The relevant data have been uploaded to the NCBI Sequence
Read Archive database. The related link is www.ncbi.nlm.nih.gov/
sra, with Submission ID SUB6925943 and BioProject ID
PRJNA604914.
Acknowledgments
This research was supported by National Key Research and Deve-
lopment Program of China (2023YFD1200300), and the earmarked
fund for CARS (CARS-34).
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary information accompanies this paper at
(https://www.maxapress.com/article/doi/10.48130/grares-0025-0009)
Dates
Received 30 December 2024; Revised 18 February 2025; Accepted
20 February 2025; Published online 9 April 2025
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