Comprehensive Analysis of Calcium Sensor Families, CBL and CIPK, in Aeluropus littoralis and Their Expression Profile in Response to Salinity

Plants have acquired sets of highly regulated and complex signaling pathways to respond to unfavorable environmental conditions during evolution. Calcium signaling, as a vital mechanism, enables plants to respond to external stimuli, including abiotic and biotic stresses, and coordinate the basic processes of growth and development. In the present study, two calcium sensor families, CBL and CIPK, were investigated in a halophyte plant, Aeluropus littoralis, with a comprehensive analysis. Here, six AlCBL genes, and twenty AlCIPK genes were studied. The analysis of the gene structure and conserved motifs, as well as physicochemical properties, showed that these genes are highly conserved during evolution. The expression levels of AlCBL genes and AlCIPK genes were evaluated under salt stress in leaf and root tissue. Based on the real-time RT-PCR results, the AlCIPK gene family had a higher variation in mRNA abundance than the AlCBL gene family. AlCIPK genes were found to have a higher abundance in leaves than in roots. The results suggest that the correlation between AlCBL genes and AlCIPK is tissue-specific, and different correlations can be expected in leaves and roots. Based on these correlations, AlCIPK3.1–AlCBL4.1 and AlCIPK1.2–AlCBL4.4 can be co-expressed in the root tissue, while AlCBL10 has the potential to be co-expressed with AlCIPK5, AlCIPK26, and AlCIPK12.3 in the leaf tissue. Our findings reveal valuable information on the structure and function of calcium sensor families in A. littoralis, a halophyte plant, that can be used in future research on the biological function of CBLs and CIPKs on salt stress resistance.


Introduction
In sessile organisms such as plants, perception and signaling of environmental stimuli are necessary for survival and growth regulation. Calcium (Ca 2+ ) is one of the signal transduction components that acts as a second messenger in all eukaryotes [1][2][3]. Ca 2+ is stored in organelles such as vacuoles, mitochondria, and endoplasmic reticulum, where abiotic stresses such as salt, cold, and drought cause a rapid increase in Ca 2+ concentration in the cytosol [3][4][5][6]. However, biotic stresses, pH dynamics, and phytohormones also can affect Ca 2+ concentration [7][8][9][10]. In addition, pollen tube development and guard cell regulation are also associated with changes in Ca 2+ concentration [8]. Calcium sensors or calcium-binding proteins recognize the modification in Ca 2+ concentrations in plant cells, and downstream pathways are induced by affecting the phosphorylation status of calcium sensors and activating protein kinases [11,12]. Calmodulin (CaM), calciumdependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) are part of the known calcium sensors in plants [13]. CBLs are plant-specific sensors that, after sensing a specific calcium signature, can physically interact with a group of protein kinases, CBLinteracting protein kinases (CIPKs), to activate downstream signaling components [14][15][16]. CBL proteins share a common helix-loop-helix structural motif (the EF-hand composition), which acts as a Ca 2+ -binding region [17]. Moreover, it seems that the EF-hand composition could affect the affinity rate of calcium ions [17].
In the plant model system Arabidopsis, diverse roles were reported for CBLs: The cbl1 mutant is very sensitive to abiotic stresses such as drought, extreme salinity, and hyperosmotic stress. Likewise, the CBL9 gene is involved in ABA signal transduction and stress-induced ABA biosynthesis pathways [18]. In addition, it was reported that CBL9 and CBL1 participate in pollen germination and flower fertilization [19]. Furthermore, it was stated that CBL1 is involved in response to aluminum stress [20] and cold stress [21,22]. Moreover, CBL7 is associated with Arabidopsis responses to alkaline stress [23]. Interestingly, it was reported that CBLs, such as CBL3 and CBL4, could modulate the potassium channel and affect potassium homeostasis [21,24]. It has also been found that the expression patterns of the CBL genes are dependent on tissues and developmental stages and the type of stress. For example, CBL1 expression is not affected by the external application of abscisic acid (ABA) but is induced in response to environmental stresses such as salt, cold, drought, and wounding [25]. While CBL2 and CBL3 do not respond to abiotic stress stimuli, they are transcriptionally induced by light stress [26]. CIPK genes also have differential expression patterns. For example, CIPK9 transcriptional regulation is more induced in response to ABA treatment and is mainly activated in shoot tissues [27]. In addition, CIPK genes in Medicago truncatula, including MtCIPK2, MtCIPK17, and MtCIPK18, were found to be upregulated in response to salinity, PEG, and ABA treatments [28]. Recently, it has been reported that a CIPK gene from chrysanthemum, CmCIPK8, could affect the expression patterns of ion transport-related genes and may enhance tolerance to salinity [29]. Moreover, CIPK10 in potatoes (StCIPK10) could increase tolerance to osmotic and drought stress by affecting the content of osmoregulation substances [30]. Additionally, it was reported that StCIPK10 can interact with several StCBLs, including StCBL4, StCBL8, StCBL1, StCBL6, StCBL12, and StCBL11 [30]. In β vulgaris, it was described that BvCIPKs are upregulated in response to NaCl treatment [31]. In Saccharum spontaneum, CIPK genes were shown to respond to abiotic stresses such as cold and water stress and ABA treatment [32]. Overall, it seems that cell signaling networks linked with CBL-CIPK play critical roles in response to abiotic stresses [33][34][35].
A. littoralis as a halophyte model can grow under high salt concentrations [36,37]. Identifying the genes related to tolerance in plants such as A. littoralis, a valuable germplasm, and determining their function can provide a better understanding of tolerance mechanisms in plants [38]. According to the mentioned materials above, the genes of CBL and CIPK families play a key role in responding to environmental stresses and regulating downstream signaling pathways, but these gene families have not been identified and investigated in A. littoralis. Here, we identified the members of CBL and CIPK families and analyzed their structure and evolution as well as their regulatory systems. In addition, the expression profiles of AlCBL and AlCIPK genes were evaluated under salinity in the root and leaf tissues of A. littoralis.

Identification of CBL and CIPK Family Genes in A. littoralis
In this study, the putative protein sequences of CBL and CIPK in rice were retrieved from the RGAP database (http://rice.plantbiology.msu.edu/, accessed on 25 December 2022) and for Arabidopsis thaliana from the TAIR database (https://www.arabidopsis.org/, accessed on 25 December 2022). Sequences were used as queries in blastp and tblastn tools, E-value < 1 × 10 −10 , to identify members of CBL and CIPK gene families from the transcriptome platform e!DAL of A. littoralis [39]. The presence of PKinase and NAF domains in CIPK proteins, as well as EF-hand domains in CBL proteins, was tested and confirmed using the CDD database [40], SMART [41], and InterPro Scan [42]. The confirmed protein sequences were renamed based on their orthologs in Arabidopsis. Further AlCBL and AlCIPK proteins were analyzed using the ExPASy online database ProtParam tool [43] to predict their physiochemical properties, including molecular weight (MW), GRAVY, and isoelectric point (pI).

Phylogenetic Analysis and Classification of AlCBL and AlCIPK Gene Families
To investigate the evolutionary relationships in calcium sensor gene families, the protein sequences of AlCBL and AlCIPK families, along with their orthologs in Arabidopsis and rice, were analyzed. First, the sequences were aligned using the ClustalW tool [44], and then a phylogenetic tree was drawn with the IQ tree software [45] using the maximum likelihood (ML) method with 1000 bootstrap replications. Finally, the tree file was restored and upgraded in the iTOL database [46].

Motif Analysis and Gene Structure of AlCBLs and AlCIPKs
Ten conserved motifs of AlCBL and AlCIPK protein sequences were predicted using the MEME motif finder [47] based on its default setting. In addition, the gene structure of AlCBL and AlCIPK genes was illustrated based on exon and intron distribution using TBtools [48].

Promoter Analysis of AlCBLs and AlCIPKs
In the current study, the upstream region, 1500 bp, of AlCBL and AlCIPK genes was analyzed using the PlantCARE tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/ html/, accessed on 25 December 2022) to identify the putative cis-regulatory elements.

Plant Materials' Growth Conditions and Salt Treatments
The cultivation of A. littoralis seeds was carried out at a temperature of 25 ± 3 and a photoperiod of 16 h of light and 8 h of darkness. Then, the cloned samples were transferred to Hoagland's solution, and after two months, salt stress treatment was started. In order to apply salinity stress, sodium chloride was gradually added; specifically, 100 mM salt was added to the solution every 3 days until the final concentration reached 600 mM. The sampling of leaf and root tissues was carried out in the time series of 0 (as a control sample), 3, 12, and 24 h after the application of salt stress. The collected samples were kept in a freezer at −80 for the next steps. All experiments were performed in three biological replications.

RNA Extraction and cDNA Synthesis
The extraction of total RNA from leaf and root tissues was carried out using a Trizol Kit (Threezol, Riragene). To remove genomic DNA from RNA, DNase I treatment (DNase I RNase-free, Thermo Scientific, Waltham, MA, USA) was applied. Finally, cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) based on the company's instructions and diluted four times.

Real-Time PCR
In the present study, the levels of mRNA abundance from six AlCBL and twelve AlCIPK genes were investigated in two tissues, roots and leaves, under salinity and normal conditions. Genes were selected based on phylogenetic analysis. The primers of candidate genes were designed using AlleleID [49] (Tables S1 and S2). The Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific) was used to evaluate the relative expression based on the manufacturer's instructions, with a Bio-Rad CFX96 machine. The temperature cycle was performed in two stages according to the manufacturer's instructions: 10 min activation stage at 95 • C, 40 cycles at 95 • C for 15 s, and 60 • C for 1 min. In the current study, three reference genes, namely AlUBQ, AlRPS3, and AlRPS3, were used for each tissue. The geometric mean of these genes was used to normalize the data. Finally, the relative expression levels of each target gene were calculated using the 2 −∆∆CT method [50].

Physicochemical Properties of AlCBLs and AlCIPKs
In the present study, six AlCBL genes and twenty AlCIPK genes were identified in the genome of A. littoralis. The evaluation of the physicochemical characteristics of CBL proteins revealed variable molecular weight in the range of 18.70 (AlCBL4.4) to 34.67 kDa (AlCBL10), and all AlCBLs were predicted as acidophilic proteins, pI less than 5.5 (Table 1). Furthermore, all AlCBLs (except for AlCBL10 protein) had negative GRAVY values, revealing that most AlCBLs have hydrophilic properties. In general, in terms of physicochemical characteristics, the AlCBL10 protein was different from other members of the AlCBL gene family, which can be more considered in molecular functional research. According to the genes' physicochemical characteristics, AlCIPK family members showed more diversity than AlCBLs. Molecular weight in AlCIPKs ranged from 42.04 (AlCIPK10.6) to 58.97 kDa (AlCIPK10.1), and pI varied from 6.21 (AlCIPK21) to 9.28 (AlCIPK10.2).

Phylogenetic Analysis of AlCBLs
AlCBL proteins, along with their orthologs in rice and Arabidopsis, were subjected to phylogenetic analysis. The results disclosed that CBL proteins could be classified into four main groups (Figure 1). None of the AlCBLs could be identified in group I. AlLAC4.1, AlLAC4.2, AlLAC4.3, and AlLAC4.4 were located in group II, AlLAC10 in group III, and AlLAC2 in group IV. In addition, AlCBLs and rice CBLs showed more similarity to each other than Arabidopsis CBLs. Overall, our results revealed that the diversity in the CBL family occurred after the splitting of monocots and dicots.
AlCBL proteins, along with their orthologs in rice and Arabidopsis, were subjected to phylogenetic analysis. The results disclosed that CBL proteins could be classified into four main groups (Figure 1). None of the AlCBLs could be identified in group I. AlLAC4.1, AlLAC4.2, AlLAC4.3, and AlLAC4.4 were located in group II, AlLAC10 in group III, and AlLAC2 in group IV. In addition, AlCBLs and rice CBLs showed more similarity to each other than Arabidopsis CBLs. Overall, our results revealed that the diversity in the CBL family occurred after the splitting of monocots and dicots.

Phylogenetic Analysis of AlCIPKs
To determine the evolutionary origin of AlCIPKs, the phylogenetic tree of AlCIPKs with their orthologs in Arabidopsis (26 CIPK proteins) and rice (33 CIPK proteins) was drawn based on protein sequences ( Figure 2). The results revealed that CIPKs could be separated into four main groups. The highest number of CIPKs was found in group III, and the lowest number was observed in group IV. Similar to AlCBLs, AlCIPKs also showed a close relationship with rice CIPKs. In addition, it can be concluded that the expansion of the CIPK family probably occurred after the derivation of monocots and dicots.

Phylogenetic Analysis of AlCIPKs
To determine the evolutionary origin of AlCIPKs, the phylogenetic tree of AlCIPKs with their orthologs in Arabidopsis (26 CIPK proteins) and rice (33 CIPK proteins) was drawn based on protein sequences (Figure 2). The results revealed that CIPKs could be separated into four main groups. The highest number of CIPKs was found in group III, and the lowest number was observed in group IV. Similar to AlCBLs, AlCIPKs also showed a close relationship with rice CIPKs. In addition, it can be concluded that the expansion of the CIPK family probably occurred after the derivation of monocots and dicots.

Gene Structure and Conserved Motifs of AlCBLs
AlCBLs with their orthologs in rice (OsCBLs) were analyzed based on their conserved motifs and domain and gene structure ( Figure 3). Ten conserved motifs were recognized in AlCBLs and OsCBLs; of those, motifs 6 and 9 were not detected in CBLs from group II, and motif 9 was only observed in OsCBLs from group III (Figure 3a). The calcium-binding superfamily, namely EF-hand 7, EF-hand 5, and EF-hand 1 domains, were observed in AlCBLs and OsCBLs, although they differed based on the location and number of domains (Figure 3b). In addition, two copies of EF-hand 7 and EF-hand 1 domains were found in AlCBL10 and its ortholog, OsCBL9, suggesting that AlCBL10 probably has more potential to interact with the downstream elements of involved pathways. In addition, AlCBLs were different based on their gene structure, and all AlCBLs had a high number of exons/introns (Figure 3c). Genes 2023, 14, x FOR PEER REVIEW 6 of 15

Gene Structure and Conserved Motifs of AlCBLs
AlCBLs with their orthologs in rice (OsCBLs) were analyzed based on their conserved motifs and domain and gene structure (Figure 3). Ten conserved motifs were recognized in AlCBLs and OsCBLs; of those, motifs 6 and 9 were not detected in CBLs from group II, and motif 9 was only observed in OsCBLs from group III (Figure 3a). The calcium-binding superfamily, namely EF-hand 7, EF-hand 5, and EF-hand 1 domains, were observed in AlCBLs and OsCBLs, although they differed based on the location and number of domains (Figure 3b). In addition, two copies of EF-hand 7 and EF-hand 1 domains were found in AlCBL10 and its ortholog, OsCBL9, suggesting that AlCBL10 probably has more potential to interact with the downstream elements of involved pathways. In addition, AlCBLs were different based on their gene structure, and all AlCBLs had a high number of exons/introns (Figure 3c).

Gene Structure and Conserved Motifs of AlCIPKs
To identify conserved motifs and determine the position of these motifs in KINAS and NAF domains, AlCIPK proteins with their orthologs in rice (OsCIPKs) were analyzed using the MEME tool (Figure 4a). The results show that the spatial distribution of the motifs in the investigated proteins is strongly conserved. All ten identified motifs were

Gene Structure and Conserved Motifs of AlCIPKs
To identify conserved motifs and determine the position of these motifs in KINAS and NAF domains, AlCIPK proteins with their orthologs in rice (OsCIPKs) were analyzed using the MEME tool (Figure 4a). The results show that the spatial distribution of the motifs in the investigated proteins is strongly conserved. All ten identified motifs were observed in AlCIPK3.1, AlCIPK3.2, AlCIPK10.1, AlCIPK10.2, AlCIPK10.4, AlCIPK20, AlCIPK23, and AlCIPK26 proteins, while motif 10 was not detectable in AlCIPK1.1, AlCIPK12.1, AlCIPK12.2, and AlCIPK12.3. Motif 5 was not detected in AlCIPK4, motif 4 was not detected in AlCIPK5, motif 1 was not observed in AlCIPK10.3, and motif 2 was not observed in AlCIPK10.5. In the AlCIPK1.2 protein, motifs 10 and 3, in the AlCIPK11 protein, motifs 10 and 6, in the AlCIPK10.

Promoter Analysis
In the present study, the upstream of AlCIPKs and AlCBLs was screened to identify the cis-regulatory elements related to stress, hormone, and growth and development ( Figure 5). The most recognized elements were related to common cis-regulatory and elements with unknown functions (Figure 5a). In addition, putative cis-regulatory elements related to transcription factors' binding site, response to phytohormones, and stresses were observed in the upstream sites of AlCIPKs and AlCBLs. The cis-regulatory elements involved in response to ABA hormone were frequently identified in the promoter sites of AlCIPKs and AlCBLs (Figure 5b). Moreover, the putative cis-regulatory

Promoter Analysis
In the present study, the upstream of AlCIPKs and AlCBLs was screened to identify the cis-regulatory elements related to stress, hormone, and growth and development ( Figure 5). The most recognized elements were related to common cis-regulatory and elements with unknown functions (Figure 5a). In addition, putative cis-regulatory elements related to transcription factors' binding site, response to phytohormones, and stresses were observed in the upstream sites of AlCIPKs and AlCBLs. The cis-regulatory elements involved in response to ABA hormone were frequently identified in the promoter sites of AlCIPKs and AlCBLs (Figure 5b). Moreover, the putative cis-regulatory elements related to GA, auxin, SA, and MeJA hormones were recognized in the upstream sites of AlCIPKs, while in AlCBLs, regulatory elements responding to GA and MeJA hormones were observed. The cis-regulatory elements involved in responsive to abiotic stresses, including low temperature, MBS, DRE, and STRE, and biotic stresses, including wound, elicitor, and defense mechanisms, were identified in the promoter regions of AlCBLs and ALCIPKs (Figure 5c). In addition, the binding sites of several TFs such as MYB, MYS, and WRKY were observed in the upstream sites of AlCIPKs and AlCBLs (Figure 5d). In general, AlCIPKs were richer than AlCBLs based on the number of stress-related cis-elements.

Expression Profiles of AlCBL Genes in Response to Salinity
The expression levels of AlCBL genes were investigated under salinity in root and leaf tissues. According to our results, AlCBL2 was not expressed under the tested conditions. It seems that AlCBL2 is not probably induced in response to salinity stress. After 3 h of salinity treatment, AlCBL4.1, AlCBL4.2, and AlCBL4.4 showed an upregulation in root tissues ( Figure 6). Three AlCBL genes, namely AlCBL4.3, AlCBL4.4, and AlCBL10, were differentially induced after 24 h; all three genes were upregulated in the leaf, while they were downregulated in the root.

Expression Profiles of AlCBL Genes in Response to Salinity
The expression levels of AlCBL genes were investigated under salinity in root and leaf tissues. According to our results, AlCBL2 was not expressed under the tested conditions. It seems that AlCBL2 is not probably induced in response to salinity stress. After 3 h of salinity treatment, AlCBL4.1, AlCBL4.2, and AlCBL4.4 showed an upregulation in root tissues ( Figure 6). Three AlCBL genes, namely AlCBL4.3, AlCBL4.4, and AlCBL10, were differentially induced after 24 h; all three genes were upregulated in the leaf, while they were downregulated in the root.

Expression Profile of AlCIPK Genes in Response to Salinity
In the root tissue, the expression levels of AlCIPK1.2 (1.96 times), AlCIPK3.1 (4.90 times), AlCIPK5 (2.32 times), AlCIPK11 (4.21 times), AlCIPK12.1 (2.62 times), and AlCIPK26 (4.63 times) were increased after three hours (h) of applying salt stress ( Figure  7). In the leaf tissue, at 3 h after applying salt stress, AlCIPK11 showed a significant modification in their expression levels after 12 hr. Interestingly, AlCIPK10.2 was notably downregulated in both root and leaf tissues. In addition, the expression levels of AlCIPK4 (3.47 times) and AlCIPK12.3 (3.78 times) were increased in leaf tissue after 24 h. In total, CIPKs were more expressed in the leaf tissue, while AlCIPK12.3 was expressed only in the leaf tissue, and AlCIPK1.1 gene expression was observed only in the root tissue. AlCIPK4, AlCIPK5, AlCIPK10.2, AlCIPK10.6, AlCIPK11, and AlCIPK12.3 genes were significantly expressed in the leaf tissue at all times of stress.

Discussion
Calcium sensors such as calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) not only participate in the processes of plant growth and development but are also involved in stress responses [30]. In the present study, the available genome of A. littoralis was used as a reference [38] and screened for the respective gene families. Six AlCBL genes and twenty AlCIPK genes were identified. Due to the importance of calcium-dependent signaling pathways, CBL and CIPK gene families have been studied in various plants. Notably, 23 CBLs and 58 CIPK genes were identified from the genome of Medicago sativa [28], 27 CIPK genes from potato [30], 9 CBLs and 30 CIPK genes from the pecan genome [51], 10 CBLs and 26 CIPKs from Arabidopsis [17], 7 CBLs and 20 CIPK genes from bread wheat [52], 7 CBLs and 23 CIPK genes from canola [53], 16 CBLs and 41 CIPK genes from quinoa [35], and 20 CIPK genes from sugar beet [31]. The different number of members of this gene family suggests that they may have been

Discussion
Calcium sensors such as calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) not only participate in the processes of plant growth and development but are also involved in stress responses [30]. In the present study, the available genome of A. littoralis was used as a reference [38] and screened for the respective gene families. Six AlCBL genes and twenty AlCIPK genes were identified. Due to the importance of calcium-dependent signaling pathways, CBL and CIPK gene families have been studied in various plants. Notably, 23 CBLs and 58 CIPK genes were identified from the genome of Medicago sativa [28], 27 CIPK genes from potato [30], 9 CBLs and 30 CIPK genes from the pecan genome [51], 10 CBLs and 26 CIPKs from Arabidopsis [17], 7 CBLs and 20 CIPK genes from bread wheat [52], 7 CBLs and 23 CIPK genes from canola [53], 16 CBLs and 41 CIPK genes from quinoa [35], and 20 CIPK genes from sugar beet [31]. The different number of members of this gene family suggests that they may have been subjected to evolutionary pressures differently in each plant [54,55]. Based on their physicochemical properties, AlCBL proteins have similar properties, except for the AlCBL10 protein. The proteins of the AlCIPK family showed more diversity. This result supports the hypothesis that AlCIPKs are highly diverse due to their involvement in different pathways [56,57]. While AlCIPKs showed a high variation in terms of gene structure, AlCIPKs could be separated into two groups. This grouping was based on their low intron number (less than three introns) and high intron number (more than ten introns). Moreover, this feature has also been reported in previous studies, where CIPKs have been classified into two groups based on their intron structure [35,58]. It was stated that partial duplication has probably affected the intron number of gene family members [59]. Moreover, it was reported that the expression levels of genes can be affected by intron number, and genes with low intron number could be faster induced [60]. According to phylogenetic analysis, both AlCBL and AlCIPK families are closely related to their rice orthologs. This finding suggests that the diversity in these gene families occurred after the derivation of monocots and dicots species [61,62].
Halophyte plants have a high potential to grow in substrates with high salinity. Therefore, these species are of great interest to investigate the mechanisms of tolerance to salinity. Such mechanisms include the absorption, transport, and homeostasis of ions, osmotic regulation, and salt removal from leaves [37,38]. Although the cultivation of these plants is not an easy task, the germplasm of halophyte plants is considered a valuable source for providing genes resistant to environmental conditions, for the implementation of plantbreeding programs [63]. In the current study, the expression profiles of AlCBLs and AlCIPKs were investigated under salt stresses in the roots and leaves of A. littoralis. AlCBLs and AlCIPKs showed tissue-specific expression patterns. For instance, AlCIPK mRNAs were more in leaves than in roots, while AlCBL4.3, AlCBL4.4, and AlCBL10 showed upregulation in roots and downregulation in shoots. This pattern might be related to the presence of as-1-specific motifs in the promoter region of AlCBL genes. Each of the AlCBL4.3, AlCBL4.1, and AlCBL2 genes had two as-1 motifs, while six as-1 motifs were observed in AlCBL4.2, three as-1 motifs were observed in AlCBL4.4, and one as-1 motif was observed in AlCBL10 promoter region.
The results revealed that the co-expression pattern of AlCBL with AlCIPK was tissuespecific, and different co-expressions were observed in two tissues of leaves and roots. Based on the expression pattern, AlCIPK3.1-AlCBL4.1 and AlCIPK1.2-AlCBL4.4 genes can be potentially co-expressed in the root tissue, while in the leaf tissue, the AlCBL10 gene can correlate with AlCIPK5, AlCIPK12.3 and AlCIPK26 genes. A positive correlation was reported between CBLs and CIPKs in response to stresses, such as salinity [64], drought [65], and disease [58]. In Arabidopsis, the interaction between CBL4 (called SOS3) and CIPK24 (called SOS2) could active the kinases and +/H+ antiporters called SOS1 and vacuolar H+-ATPase to increase stress tolerance [53,66,67]. Subsequent research in Arabidopsis showed that the AtCBL10 gene also interacts with AtCIPK24. Thus, the CBL10-CIPK24 complex interacts with vacuoles to protect the shoot from damage caused by salt stress [67]. This result suggests that calcium sensors may exhibit very different functions despite high sequence similarity or close evolutionary relationships.

Conclusions
This review is the first comprehensive study of the family of calcium sensors with the aim of clarifying the evolution, expression patterns, and possible functions of the genes of this superfamily in A. littoralis in response to salinity stress. These findings provide information to predict the function of calcium sensor genes in plant tolerance to salinity stress. Additional studies on the expression of AlCBL and AlCIPK family genes under different abiotic stresses in future research can be useful in understanding the mechanism of gene expression adjustments related to the SOS pathway. The AlCIPK genes reported in this research, while providing preliminary information, provide a basis for identifying the