Molecular characterization of functional domains in the protein kinase SOS2 that is required for plant salt tolerance

The SOS3 (for SALT OVERLY SENSITIVE3) calcium binding protein and SOS2 protein kinase are required for sodium and potassium ion homeostasis and salt tolerance in Arabidopsis. We have shown previously that SOS3 interacts with and activates the SOS2 protein kinase. We report here the identification of a SOS3 binding motif in SOS2 that also serves as the kinase autoinhibitory domain. Yeast two-hybrid assays as well as in vitro binding assays revealed a 21-amino acid motif in the regulatory domain of SOS2 that is necessary and sufficient for interaction with SOS3. Database searches revealed a large family of SOS2-like protein kinases containing such a SOS3 binding motif. Using a yeast two-hybrid system, we show that these SOS2-like kinases interact with members of the SOS3 family of calcium binding proteins. Two-hybrid assays also revealed interaction between the N-terminal kinase domain and the C-terminal regulatory domain within SOS2, suggesting that the regulatory domain may inhibit kinase activity by blocking substrate access to the catalytic site. Removal of the regulatory domain of SOS2, including the SOS3 binding motif, resulted in constitutive activation of the protein kinase, indicating that the SOS3 binding motif can serve as a kinase autoinhibitory domain. Constitutively active SOS2 that is SOS3 independent also was produced by changing Thr(168) to Asp in the activation loop of the SOS2 kinase domain. Combining the Thr(168)-to-Asp mutation with the autoinhibitory domain deletion created a superactive SOS2 kinase. These results provide insights into regulation of the kinase activities of SOS2 and the SOS2 family of protein kinases.     

Photoperiodic flowering occurs under internal and external coincidence

Determining the proper time to flower is important to ensure the reproductive success of plants. The model plant Arabidopsis is able to measure day-length and promotes flowering in long day (LD) conditions. One of the most prominent mechanisms in photoperiodic flowering is the clock-regulated gene expression of CONSTANS (CO) and the stabilization and activation of CO protein by light (regarded as external coincidence). We recently demonstrated that timing of the blue-light dependent formation of FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and GIGANTEA (GI) protein complex is crucial for regulating the timing of CO gene expression. The expression of FKF1 and GI is clock regulated, and their expression patterns have the same phase in LD (regarded as internal coincidence) but not in short day (SD) conditions, where floral induction is greatly delayed. Hence, timing of the FKF1-GI complex formation is regulated by the coincidence of both external and internal cues. Here, we propose a molecular mechanism for CO regulation by FKF1-GI complex formation.Key words: Arabidopsis, circadian clock, photoperiodic flowering, CONSTANS, GIGANTEA, FKF1, CDF1     

A Calcium Sensor-Regulated Protein Kinase,CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, Is Required for Pollen Tube Growth and Polarity

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca²⁺ throughout the bulging tip, whereas LaCl₃, a Ca²⁺ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca²⁺ homeostasis through its potential function in the modulation of Ca²⁺ influx.In flowering plants, fertilization is mediated by pollen tubes that extend directionally toward the ovule for sperm delivery (Krichevsky et al., 2007; Johnson, 2012). The formation of these elongated tubular structures is dependent on extreme polar growth (termed tip growth), in which cell expansion occurs exclusively in the very apical area (Yang, 2008; Rounds and Bezanilla, 2013). As this type of tip growth is amenable to genetic manipulation and cell biological analysis, the pollen tube is an excellent model system for the functional analysis of essential genes involved in polarity control and fertilization (Yang, 2008; Qin and Yang, 2011; Bloch and Yalovsky, 2013).It is well established that Ca²⁺ plays a critical role in pollen germination and tube growth (Konrad et al., 2011; Hepler et al., 2012). A steep tip-focused Ca²⁺ gradient has been detected at the tip of elongating pollen tubes (Rathore et al., 1991; Pierson et al., 1994; Hepler, 1997). In previous studies, artificial dissipation of the Ca²⁺ gradient seriously inhibited tip growth of pollen tubes, whereas elevation of internal Ca²⁺ level induced bending of the growth axis toward the zone of higher Ca²⁺. These studies suggest that Ca²⁺ not only controls pollen tube elongation but also modulates growth orientation (Miller et al., 1992; Malho et al., 1994; Malho and Trewavas, 1996; Hepler, 1997). These Ca²⁺ signatures are perceived and relayed to downstream responses by a complex toolkit of Ca²⁺-binding proteins that function as Ca²⁺ sensors (Yang and Poovaiah, 2003; Harper et al., 2004; Dodd et al., 2010).To date, four major Ca²⁺ sensor families have been identified in Arabidopsis (Arabidopsis thaliana), including calcium-dependent protein kinase, calmodulin (CaM), calmodulin-like (CML), and CALCINEURIN B-LIKE (CBL) proteins (Luan et al., 2002, 2009; Yang and Poovaiah, 2003; Harper et al., 2004). Calcium-dependent protein kinase family members comprise a kinase domain and a CaM-like domain in a single protein; thus, they act not only as a Ca²⁺ sensor but also as an effector, designated as sensor responders (Cheng et al., 2002). In contrast, CaM, CML, and CBL proteins do not have any enzymatic domains but transmit Ca²⁺ signals to downstream targets via Ca²⁺-dependent protein-protein interactions. Therefore, they have been designated as sensor relays (McCormack et al., 2005). While CaM and CML proteins interact with a diverse array of target proteins, it is generally accepted that CBLs interact specifically with a group of Ser/Thr protein kinases termed CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASEs (CIPKs; Luan et al., 2002; Kolukisaoglu et al., 2004).In Arabidopsis, several CBLs coupled with their target CIPKs have been demonstrated to function in the regulation of ion homeostasis and stress responses (Luan et al., 2009). Under salt stress, SALT OVERLY SENSITIVE3 (SOS3)/CBL4-SOS2/CIPK24 regulate SOS1 at the plasma membrane for Na⁺ exclusion, whereas CBL10-CIPK24 complexes appear to regulate Na⁺ sequestration at the tonoplast (Liu et al., 2000; Qiu et al., 2002; Kim et al., 2007; Quan et al., 2007). For low-K⁺ stress, CBL1 and CBL9, with 87% amino acid sequence identity, interact with CIPK23, which regulates a voltage-gated ion channel (ARABIDOPSIS K⁺ TRANSPORTER1) to mediate the uptake of K⁺ in root hairs (Li et al., 2006; Xu et al., 2006; Cheong et al., 2007). In addition, CBL1 integrates plant responses to cold, drought, salinity, and hyperosmotic stresses (Albrecht et al., 2003; Cheong et al., 2003), and CBL9 is involved in abscisic acid signaling and biosynthesis during seed germination (Pandey et al., 2004). Over the past decade, the functions of CBL-CIPK complexes in abiotic stress tolerance have been studied extensively, but only limited studies focus on CBL family members in pollen tube growth. For example, CBL3 overexpression caused a defective phenotype in pollen tube growth (Zhou et al., 2009). Overexpression of CBL1 or its closest homolog CBL9 inhibited pollen germination and perturbed tube growth at high external K⁺, whereas disruption of CBL1 and CBL9 leads to a significantly reduced growth rate of pollen tubes under low-K⁺ conditions (Mähs et al., 2013). The potential roles of CIPKs in pollen tubes so far appear to be completely unknown.In this study, we demonstrated that Arabidopsis CIPK19, a CIPK specifically expressed in pollen grains and pollen tubes, functions in pollen tube tip growth, providing a new insight into the function of the CBL-CIPK network in the control of growth polarity during pollen tube extension in fertilization.     

A role for GIGANTEA: Keeping the balance between flowering and salinity stress tolerance

The initiation of flowering in Arabidopsis is retarded or abolished by environmental stresses. Focusing on salt stress, we provide a molecular explanation for this well-known fact. A protein complex consisting of GI, a clock component important for flowering and SOS2, a kinase activating the [Na⁺] antiporter SOS1, exists under no stress conditions. GI prevents SOS2 from activating SOS1. In the presence of NaCl, the SOS2/GI complex disintegrates and GI is degraded. SO2, together with the Ca²⁺-activated sensor of sodium ions, SOS3, activates SOS1. In gi mutants, SOS1 is constitutively activated and gi plants are more highly salt tolerant than wild type Arabidopsis. The model shows GI as a transitory regulator of SOS pathway activity whose presence or amount connects flowering to environmental conditions.     

The Suppression of WRKY44 by GIGANTEA-miR172 Pathway Is Involved in Drought Response of Arabidopsis thaliana

Water availability is an important environmental factor that controls flowering time. Many plants accelerate flowering under drought conditions, a phenomenon called drought escape. Four pathways are involved in controlling flowering time, but which ones participate in drought escape is not yet known. In this study, plants with loss-of-function mutations of GIGANTEA (GI) and CONSTANS (CO) exhibited abnormal drought-escape phenotypes. The peak mRNA levels of GI and FKF1 (Flavin-binding Kelch domain F box protein 1) and the mRNA levels of CO and FT (Flowering locus T) changed under drought stress. The microRNA factor miRNA172E was up-regulated by drought stress, and its up-regulation was dependent on GI, while other miRNA172s were not. Water-loss analyses indicated that gi mutants were more sensitive while miRNA172 over-expressing (miRNA172-OX) plants were less so to drought stress than wild-type plants. Digital gene expression and real-time PCR analyses showed that WRKY44 was down-regulated by GI and miRNA172. The WRKY44 protein could interact with TOE1 (a target of miRNA172) in a yeast two-hybrid system. We proposed that GI–miRNA172–WRKY44 may regulate drought escape and drought tolerance by affecting sugar signaling in Arabidopsis.     

Co-expression of the Arabidopsis SOS genes enhances salt tolerance in transgenic tall fescue (Festuca arundinacea Schreb.)

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. Here, we report that overexpression of Arabidopsis thaliana SOS1+SOS2+SOS3 genes enhanced salt tolerance in tall fescue. The transgenic plants displayed superior growth and accumulated less Na⁺ and more K⁺ in roots after 350 mM NaCl treatment. Moreover, Na⁺ enflux, K⁺ influx, and Ca²⁺ influx were higher in the transgenic plants than in the wild-type plants. The activities of the enzyme superoxide dismutase, peroxidase, catalase, and proline content in the transgenic plants were significantly increased; however, the malondialdehyde content decreased in transgenic plants compared to the controls. These results suggested that co-expression of A. thaliana SOS1+SOS2+SOS3 genes enhanced the salt tolerance in transgenic tall fescue.     

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.     

SCABP8/CBL10, a putative calcium sensor, interacts with the protein kinase SOS2 to protect Arabidopsis shoots from salt stress

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.     

Comprehensive structural,interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice

Calcium ion is involved in diverse physiological and developmental pathways. One of the important roles of calcium is a signaling messenger, which regulates signal transduction in plants. CBL (calcineurin B-like protein) is one of the calcium sensors that specifically interact with a family of serine–threonine protein kinases designated as CBL-interacting protein kinases (CIPKs). The coordination of these two gene families defines complexity of the signaling networks in several stimulus-response-coupling during various environmental stresses. In Arabidopsis, both of these gene families have been extensively studied. To understand in-depth mechanistic interplay of CBL–CIPK mediated signaling pathways, expression analysis of entire set of CBL and CIPK genes in rice genome under three abiotic stresses (salt, cold and drought) and different developmental stages (3-vegetative stages and 11-reproductive stages) were done using microarray expression data. Interestingly, expression analysis showed that rice CBLs and CIPKs are not only involved in the abiotic stress but their significant role is also speculated in the developmental processes. Chromosomal localization of rice CBL and CIPK genes reveals that only OsCBL7 and OsCBL8 shows tandem duplication among CBLs whereas CIPKs were evolved by many tandem as well as segmental duplications. Duplicated OsCIPK genes showed variable expression pattern indicating the role of gene duplication in the extension and functional diversification of CIPK gene family in rice. Arabidopsis SOS3/CBL4 related genes in rice (OsCBL4, OsCBL5, OsCBL7 and OsCBL8) were employed for interaction studies with rice and Arabidopsis CIPKs. OsCBLs and OsCIPKs are not only found structurally similar but likely to be functionally equivalent to Arabidopsis CBLs and CIPKs genes since SOS3/CBL4 related OsCBLs interact with more or less similarly to rice and Arabidopsis CIPKs and exhibited an interaction pattern comparable with Arabidopsis SOS3/CBL4.     

Poplar calcineurin B‐like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS2 in the vacuolar membrane

The calcineurin B‐like protein (CBL) family represents a unique group of calcium sensors in plants. In Arabidopsis, CBL10 functions as a shoot‐specific regulator in salt tolerance. We have identified two CBL10 homologs, PtCBL10A and PtCBL10B, from the poplar (Populus trichocarpa) genome. While PtCBL10A was ubiquitously expressed at low levels, PtCBL10B was preferentially expressed in the green‐aerial tissues of poplar. Both PtCBL10A and PtCBL10B were targeted to the tonoplast and expression of either one in the Arabidopsis cbl10 mutant could rescue its shoot salt‐sensitive phenotype. Like PtSOS3, both PtCBL10s physically interacted with the salt‐tolerance component PtSOS2. But in contrast to the SOS3‐SOS2 complex at the plasma membrane, the PtCBL10‐SOS2 interaction was primarily associated with vacuolar compartments. Furthermore, overexpression of either PtCBL10A or PtCBL10B conferred salt tolerance on transgenic poplar plants by maintaining ion homeostasis in shoot tissues under salinity stress. These results not only suggest a crucial role of PtCBL10s in shoot responses to salt toxicity in poplar, but also provide a molecular basis for genetic engineering of salt‐tolerant tree species.     

A Calcium-Independent Activation of the Arabidopsis SOS2-Like Protein Kinase24 by Its Interacting SOS3-Like Calcium Binding Protein1

The salt stress-induced SALT-OVERLY-SENSITIVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) involves the perception of a calcium signal by the SOS3 and SOS3-like CALCIUM-BINDING PROTEIN8 (SCaBP8) calcium sensors, which then interact with and activate the SOS2 protein kinase, forming a complex at the plasma membrane that activates the SOS1 Na⁺/H⁺ exchanger. It has recently been reported that phosphorylation of SCaBP proteins by SOS2-like protein kinases (PKSs) stabilizes the interaction between the two proteins as part of a regulatory mechanism that was thought to be common to all SCaBP and PKS proteins. Here, we report the calcium-independent activation of PKS24 by SCaBP1 and show that activation is dependent on interaction of PKS24 with the C-terminal tail of SCaBP1. However, unlike what has been found for other PKS-SCaBP pairs, multiple amino acids in SCaBP1 are phosphorylated by PKS24, and this phosphorylation is dependent on the interaction of the proteins through the PKS24 FISL motif and on the efficient activation of PKS24 by the C-terminal tail of SCaBP1. In addition, we show that Thr-211 and Thr-212, which are not common phosphorylation sites in the conserved PFPF motif found in most SCaBP proteins, are important for this activation. Finally, we also found that SCaBP1-regulated PKS24 kinase activity is important for inactivating the Arabidopsis plasma membrane proton-translocating adenosine triphosphatase. Together, these results suggest the existence of a novel SCaBP-PKS regulatory mechanism in plants.Calcium is a ubiquitous second messenger that plays an important role in the regulation of plant growth and development. Many different types of calcium-binding proteins have been identified in plants (Harper et al., 2004), including the SALT-OVERLY-SENSITIVE3 (SOS3)-LIKE CALCIUM BINDING PROTEINS (SCaBPs; Liu and Zhu, 1998; Gong et al., 2004). Because the calcium-binding domain of these proteins shares sequence similarity with the yeast calcineurin B subunit, they have also been called CALCINEURIN B-LIKE PROTEINS (CBLs; Kudla et al., 1999; Luan et al., 2002). The founding member of this gene family, SOS3, was identified in a genetic screen from a salt-sensitive Arabidopsis (Arabidopsis thaliana) mutant (Liu and Zhu, 1998). SCaBP/CBL proteins interact with the SOS2-LIKE PROTEIN KINASES (PKSs)/CBL-INTERACTING PROTEIN KINASES (CIPKs; Shi et al., 1999; Halfter et al., 2000; Guo et al., 2001). The genetic linkage between these two families was established after identification of SOS2 from a genetic screen similar to the one that identified the sos3 mutant (Liu et al., 2000). SOS3 interacts with SOS2 in vivo and in vitro and activates SOS2 in a calcium-dependent manner in vitro (Halfter et al., 2000). The SOS3-SOS2 complex further activates SOS1, a plasma membrane (PM) Na⁺/H⁺ antiporter, by directly phosphorylating the SOS1 C terminus (Shi et al., 2000; Qiu et al., 2002; Quintero et al., 2002, 2011; Yu et al., 2010).In addition to the calcium-dependent activation of PKSs by SCaBP calcium sensors, two other regulatory mechanisms have been identified for these protein families. First, PKSs have a conserved 21-amino acid peptide (FISL motif) in their regulatory domain that is necessary for efficient interaction with the SCaBP calcium sensors (Guo et al., 2001; Albrecht et al., 2001; Gong et al., 2004). The PKS regulatory domain interacts with its kinase domain via the FISL motif to repress PKS activity; interaction of SCaBP with the PKS FISL motif releases the kinase domain inhibition allowing for kinase activity (Guo et al., 2001; Gong et al., 2004). Second, the PKSs phosphorylate a Ser residue in the conserved C-terminal PFPF motif of the SCaBP proteins. This phosphorylation enhances the interaction between the two proteins and fully activates the complex (Lin et al., 2009; Du et al., 2011; Hashimoto et al., 2012).In this study, we identified a novel PKS activation mechanism involving the calcium-independent activation of PKS24 by SCaBP1 and show that it requires binding of SCaBP1 to the FISL motif of PKS24 and the involvement of two Thr residues in the SCaBP1 C-terminal tail.     

Phosphorylation of SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 Protein Kinase Stabilizes Their Protein Complex and Regulates Salt Tolerance in Arabidopsis

The Salt Overly Sensitive (SOS) pathway plays an important role in the regulation of Na⁺/K⁺ ion homeostasis and salt tolerance in Arabidopsis thaliana. Previously, we reported that the calcium binding proteins SOS3 and SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCaBP8) nonredundantly activate the protein kinase SOS2. Here, we show that SOS2 phosphorylates SCaBP8 at its C terminus but does not phosphorylate SOS3. In vitro, SOS2 phosphorylation of SCaBP8 was enhanced by the bimolecular interaction of SOS2 and SCaBP8 and did not require calcium ions. In vivo, this phosphorylation was induced by salt stress, occurred at the membrane, stabilized the SCaBP8-SOS2 interaction, and enhanced plasma membrane Na⁺/H⁺ exchange activity. When a Ser at position 237 in the SCaBP8 protein (the SOS2 phosphorylation target) was mutated to Ala, SCaBP8 was no longer phosphorylated by SOS2 and the mutant protein could not fully rescue the salt-sensitive phenotype of the scabp8 mutant. By contrast, when Ser-237 was mutated to Asp to mimic the charge of a phosphorylated Ser residue, the mutant protein rescued the scabp8 salt sensitivity. These data demonstrate that calcium sensor phosphorylation is a critical component of SOS pathway regulation of salt tolerance in Arabidopsis.     

Growth,ionic response,and gene expression of shoots and roots of perennial ryegrass under salinity stress

Growth, ionic responses, and expression of candidate genes to salinity stress were examined in two perennial ryegrass accessions differing in salinity tolerance. The salinity tolerant (PI265349) and sensitive accessions (PI231595) were subjected to 75-mM NaCl for 14 days in a growth chamber. Across two accessions, salinity stress increased shoot dry weight and concentrations of malondialdehyde (MDA) and Na⁺ in the shoots and roots, but decreased shoot Ca²⁺ and root K⁺ concentrations. Salinity stress also increased root expressions of SOS1, PIP1, and TIP1. Plant height and chlorophyll content were unaffected by salinity stress in the tolerant accession but significantly decreased in the sensitive accession. Shoot MDA content did not change in the tolerant accession but increased in the sensitive accession. A more dramatic increase in Na⁺ was found in the roots of the sensitive accession. Relative to the control, salinity stress reduced expression of SOS1, NHX1, PIP1, and TIP1 in the shoots but increased expression of these genes in the roots of the tolerant accession. Expression levels of SOS1 increased in the roots and expression of NHX1 increased in the shoots but decreased in the roots of the sensitive accession under salinity stress. A decline in PIP1 expression in the shoots and dramatic increases in TIP expression in both shoots and roots were found in the sensitive accession under salinity stress. The results suggested maintenance of plant growth and leaf chlorophyll content, lesser Na⁺ accumulation in the roots, and lower lipid peroxidation in the shoots which could be associated with salinity tolerance. The decreased expressions of SOS1, NHX1, and TIP1 in the shoots, and increased expressions of NHX1 and PIP1 in the roots might also be related to salinity tolerance in perennial ryegrass.