Plants utilize many various strategies to tolerate salinity conditions. Plant requires
a high cytosolic K+/Na+ ratio in the cytoplasm to survive under salt stress. Previous studies in various
crop species such as barley, rice and maize indicated that the limiting of Na+ accumulation or the ability to maintain K+ in the shoot under salinity has been positively correlated with salt tolerance [16,
89]. Under salinity stress, some tolerance mechanisms are evolved in plants to regulate
K+ and Na+ leading cellular homeostasis under salinity stress. Restriction of Na+ uptake, Na+ exclusion to the soil and compartmentation of Na+ from cytosol to vacuoles and control of Na+ loading in xylem are crucial mechanisms for tolerance to salinity [57, 82]. Many of these salt tolerance
mechanisms depend on H+, K+ and Na+ transporters, such as SOS1, HKT, HAK and NHX to maintain the cellular ionic homeostasis
for salt tolerance .
Salinity stress reduces the soil water potential, thus induces water deficit and rapidly
transmits a water deficit signal from root to shoot and finally contributes intracellular
turgor reduction . Therefore, salinity causes a reduction in stomatal conductance
that is the most significant response that immediately occurs after plant exposure
to salt. Due to stomatal closure, salinity stress reduces the rate of transpiration
(water loss) limiting a load of toxic ions within the transpiration stream in plant,
and photosynthesis (CO2 uptake), thus inhibits carbon fixation and the accumulation
of the ions in the shoot part of the plant affecting plant growth [43, 65, 92]. These
events have been found in several plant species [10, 30]. Koyro  showed that a
decrease in stomatal conductance is an adaptive mechanism to salinity via maintaining
salts at subtoxic levels.
Cytosolic calcium is a second messenger in signalling of abiotic stress such as salinity.
The initial increase in cytosol Ca2+ could be due to the production of ROS (mainly H2O2) sourced by the membrane NADPH oxidases (encoded by RBOH) are involved in signaling
during stress . The results of this study showed that the gene expression of Rboh was significantly up-regulated in the root and shoot samples of salt-tolerant mutant
genotype in compared to its wild-type genotype in the short term (six hours) after
exposure to salt stress. Moreover, ROS increase the cytosol Ca2+ concentration resulting in the regulation of ion homeostasis via the activity of
SOS1 antiporter, MAP kinase, SOS2 and CBLs . According to the overexpression
of these genes in the salt-tolerant mutant genotype at the same time of six hours,
these events were observed in the present and the previous study .
Drerup et al,  demonstrated that RBOH in Arabidopsiscan be activated by CIPK26/CBL1/9. In this study, the parallel up-regulation of these
was shown in the salt-tolerant mutant genotype at six hours after exposure to the
salinity in comparison to its wild-type genotype. ROS function as important signaling
molecules in adaptive and developmental processes of plants to abiotic and biotic
stresses that can activate several signal pathways, such as hormonal signaling networks
to enhance the salinity tolerance . In addition, ROS activate K+ ion channels leading to retain cellular homeostasis [26, 28].
Chung et al,  showed that the activity of salt stress-induced SOS1transcript requires RBOH through increasing of Ca2+ concentration in the cytosol. Ca2+ is sensed by CBL4 (SOS3) protein which after interaction with the serine/threonine
protein kinase CIPK24 (SOS2) produces the SOS3/SOS2 complex. This complex activates
the plasma membrane Na+/H+ antiporter SOS1 for Na+ efflux from cell to control ion homeostasis [75, 97, 101]. On the other hand, SOS3/SOS2
complex induces the activity of ion transporters such as NHX1 to compartmentalize
Na+ from the cytoplasm to the vacuoles .The driving force for this transporter is
provided by two main vacuolar H+-pumps; H+-ATPase and H+-pyrophosphatase (V-PPase) indicating those have essential roles in response to salt
conditions [29, 37]. In our previous study, the overexpression of the parallel genes
(SOS1-3) in SOS pathway, NHX1 transporter and HVA pump were observed in the salt-tolerant mutant genotype in contrast with wild-type
genotype at 6 h after exposure to salinity stress .
Under salt stress, NADPH oxidase leading to the generation of ROS results in MAP
Kinase activity that linked to salt tolerance . MAPK superfamily is as a part of the serine/threonine kinases, is a key player in
some of the critical roles in plant signaling networks and is tolerant to various
stresses including drought and salinity . Moreover, many studies have indicated that in addition to MAPK pathway activity,
salinity stress also induces hormones production and signals, such as ethylene in
plants . The plant hormones such as ethylene have a major role in plant development,
throughout germination, growth, and response to stress conditions. Previous studies have indicated ROS are essential for transduction of ethylene signal
in the regulation of Na+ and K+ homeostasis to initiate the tolerance . Moreover, it is shown that salt
tolerance was induced by ethylene in Arabidopsis [13, 94]. Ethylene production is
as a mediator component in the response to stress conditions in barley. To produce ethylene, S-adenosyl-L-methionine is converted to ACC by ACC synthase (or
ACS). Finally, ACC is modified by ACC oxidase (or ACO), which can influence the expression of another set of genes . Salt and osmotic stress induced the conversion of ACC to ethylene in the halophyte
Allenrolfea occidentalis . Ethylene binding to the receptor which interacts with Constitutive Response
(CTR), initiates a transcriptional cascade and downstream ethylene responses .
Li et al,  demonstrated that ethylene production and activity of ACO were significantly increased
in cucumber seedlings under salinity stress (75 mM NaCl). Moreover, the ethylene-responsive
element binding factor (ERF) was vital in cotton under stress conditions . Overexpression
of sugarcane and soybean ERFs in tobacco conferred tolerance to high salinity stress
(200 mM) . Regarding parallel overexpression of these genes at hormonal pathway networks
of ethylene production, these mechanisms were observed in the salt-tolerant mutant
genotype after six hours of exposure to the salt stress to enhance the salinity tolerance.
Moreover, potassium (K+) is an essential factor in osmotic processes for resistance to salinity and drought.
K+ decreases the toxic effects of Na+ and maintains high K+/ Na+ ratio in shoots, especially in leaves, which is important in glycophytes for more
tolerance to salinity conditions . On the other hand, the production of ROS depends
on K+ availability. Reduction in K+ content is associated with increased enzymes activity involved in the detoxification
of H2O2. Therefore, the increase in K+ content reduces ROS using limiting the membrane NADPH oxidase resulting in salinity
tolerance . K+ channels are vital to maintain and support the development and plant growth. Researches categorized six gene families, including three channel families and three
transporter families (HAK/KUP/KT, HKT, and CPA) [31, 85].
The main transporter of salt stress tolerance is HKT antiporter [65, 78]. HKT transporters
have two Sub-families that sub-family1 transporters are only permeable to Na+, while sub-family2 transport both K+ and Na+ . HKT1;5is a Na+ selective transporter, expressed in the plasma membrane of parenchyma cells surrounding
xylem vessels [4, 67]. HKTs are involved in Na+ long-distance translocation by contributing to Na+ unloading through the xylem, preventing the large accumulation of Na+ in leaves . The up-regulation of the HKT1 transporters activity causes a decrease in leaf Na+ content [25, 73]. Therefore, it increases the ability of control of K+/Na+ homeostasis for salinity tolerance . It is revealed that OsHKT1;5 reduced the root to shoot delivery of Na+ and increased salinity tolerance in rice [36, 64, 77]. In the present study, the
gene expression of HKT1;5 was up-regulated at 6 h after exposure to salt stress in
the salt-tolerant mutant genotype. In general, in parallel of increase in Rboh gene expression, the up-regulated genes of MAPK, ACC synthase, HAK, HVP and HKT were observed at the same time point (6 h) of exposure to salt stress in the salt-tolerant
mutant genotype in comparison to the wild type genotype that was shown in Fig 2.
Furthermore, ethylene causes the preservation of cellular K+ via an increase in transcript level of AtHAK transporter in Arabidopsis under salt stress . Under salt stress, HAK transporter plays a key role in K+ uptake through the root, and K+ long-distance transport through loading and unloading in the vascular tissue [79,
87]. Shin and Schachtman (2004) revealed that HAK5 expression gene in Arabidopsisroot depended on RBOH activity and ROS. Moreover, HAK5 activity in Arabidopsisroots is regulated by CIPK23, CBL1, CBL8, CBL9, and CBL10 proteins [76, 97]. In the
current research, an increase in gene expression of HAK in the root and shoot samples of the salt-tolerant mutant genotype in addition to
six hours, was considerably observed at 24 hours and 48 hours in the root sample of
that after exposure to salt stress in contrast to its wild-type genotype.
TPK1/KCO1 channel, another important family of K+ channel have been localized in the vacuolar membrane. This channel contains the binding
sites for Ca2+ and 14-3-3 proteins involved in the K+ transport from vacuole to cytosol to contribute a favorable Na+/K+ ratio and ion homeostasis [85, 97]. In the present study, the transcript level of TPK1/KCO1 gene and 14-3-3
protein was up-regulated after 6 h of exposure to salinity in the salt-tolerant mutant
genotype in comparison with its wild type genotype.
Salt stress causes the ROS production inducing some enzymatic antioxidants to remove them for salinity tolerance . In this research,
the higher expression of peroxidase was observed to remove H2O2 content in the salt-tolerant mutant genotype in comparison to its wild-type genotype
after 6 h of exposure to salinity. Under salt stress, APX is one of the most significant
antioxidant enzymes in plant cells and plays an essential role in the control of ROS
levels regulated by redox signals and H2O2 . Many researchers reported an increase in APX activity in response to abiotic
stresses such as salinity, drought, chilling, and metal toxicity [32, 55]. Moreover, APX has a much higher affinity for H2O2 than CAT, making efficient scavengers of H2O2 with high concentration under stress conditions . Therefore, due to more accumulation
of H2O2 in the wild-type genotype, the genes expression of enzymatic antioxidants, such as
CAT, POX, and APX for ROS scavengers were significantly up-regulated in contrast to
salt-tolerant mutant genotype in the short term of six hours after exposure to the
salt stress. The results of this study were similar to the results of Kiani et al,
. Furthermore, because of stomatal closure, the rate of evaporation via transpiration
stream and H2O2 production via photosystems (I and II) were reduced in plants, which is an important
mechanism of salt tolerance [7, 65].
Many researchers indicated that salt stress induces MAPK pathway, also activation
of transcription factors, such as WRKY in Arabidopsis . Transcription factors
are the most important regulators that control a wide range of gene expressions in
different signaling pathways through binding to the specific cis-acting element in
the promoters of genes . Among all transcription factors, bZIP, WRKY, MYB, CTR/DRE,
AP2, NAC, C2H2 zinc finger gene, and DREB families observing high expression levels,
comprise a large number of stress-responsive members [42,
59]. In this research, the results of mRNA-seq analysis revealed that the expression
levels of important transcription factors were increased in the salt-tolerant mutant
genotype in comparison to its wild-type genotype at six hours after exposure to the
salinity stress that was included WRKY, ERF, bZIP, AP2/ERF, NAC, AP2/EREBP, Cytochrome P450, CTR/DRE, MIKC, MAD, and HSF. Many researchers demonstrated
that transcription factors, such as WRKY and NAC have main roles in ROS signaling
pathways in response to stresses resulting in salt tolerance in Arabidopsis [15, 51].
Nakashima et al,  revealed that the upregulation of a NAC in both rice and wheat
plays a vital role in salt tolerance. Moreover,
Song et al,  demonstrated that NAC overexpression induced by ROS (H2O2) in rice under salinity
stress, which may regulate the synthesis and accumulation of components, such as proline,
sugar, and LEA proteins that play the important roles in tolerance to stress.
The RNA-seq, as high-throughput sequencing of cDNA is the most important powerful
application for analysis during these decades. Recently, a genome sequence and transcript
profiling of barley has been reported for the stress-responsive genes [9, 93]. In this research, sequencing of the cDNA samples of the salt-tolerant mutant and its
wild-type genotypes in the early time point (six hours) after exposure to the salt stress (300 mM NaCl treatment) yielded about 20 million reads for each genotype.
Moreover, a total number of differential expression transcripts included 7116, 1586, and 1479 DE transcripts of which with significant overexpression were
obtained in the salt-tolerant mutant and its wild-type genotypes, respectively.
Many types of research demonstrated that the tolerant plants displayed a decreased
respiratory rate, whereas sensitive plants displayed an increased respiratory rate.
The certain halophytes decrease respiration under salt stress, due to deploying their
carbon reserves in the shoot for reproducing and maintaining tissue tolerance [12,
38]. Moreover, Jacoby et al,  suggested that the tolerant varieties to salt stress
allocate less of its fixed carbon into respiration, and more into growth. According
to the up-regulation of some genes related to respiration pathway including glycolysis,
Krebs cycle, citric acid cycle, and the electron transport chain in mitochondria in
the wild type genotype, RNA-seq analysis data was detected that the respiratory rate
was higher in this genotype when compared to the salt-tolerant mutant genotype. Therefore, instead of maintaining the plant tissues,
the stored energy and carbon was consumed in the wild-type genotype at an early time point under salinity stress.