Calcium (Ca²⁺), whose level fluctuates when cells undergo external changes, acts as a universal secondary messenger for numerous signals and confers specific cellular responses (Ma L. et al., 2019). Ca²⁺ sensors or Ca²⁺-binding proteins can sense changes in cytoplasmic Ca²⁺ concentrations and transmit signals to regulate the function of the downstream proteins (such as transcriptional factors or membrane transporters) and elicit changes in cellular processes (such as gene expression or ionic fluxes) in response to environmental changes (Wang et al., 2020). In plants, there are three major classes of Ca²⁺-binding proteins include calmodulin (CaM) and CaM-like proteins (CMLs), calcineurin B-like proteins (CBLs), and calcium-dependent protein kinases (CDPKs) (Tang et al., 2020).

CBL proteins contain four typical helix-loop-helix motifs (EF-hands) for calcium-binding. However, unlike CDPKs, CBL proteins lack kinase activity and function in calcium signal transduction by interacting specifically with a group of Ser/Thr protein kinases, namely CBL-interacting protein kinases (CIPKs)/SNF1-related kinase group 3 (SnRK3s) (Hrabak et al., 2003). After sensing the change of Ca²⁺ level, CBLs physically interact with the NAF/FISL motifs at the C-terminal of CIPKs to activate CIPKs, and then, the activated CIPKs participate in calcium signaling by phosphorylating target proteins (Luan, 2009). Multiple experiments have proved the core role of the CBL-CIPK signal pathway in fine-tuning plants adaptive response to external environmental changes, including Arabidopsis, rice (Kolukisaoglu et al., 2004; Kanwar et al., 2014), maize (Chen et al., 2011), grapevine (Xi et al., 2017), turnip (Yin et al., 2017), eggplant (Li et al., 2016), and pineapple (Aslam et al., 2019). Among the 10 CBLs and 26 CIPKs in Arabidopsis, not only does each CBL interact with several CIPKs, but each CIPK interacts with one or more CBLs. Such interaction specificity and overlap between different members of CBL and CIPK family may confer both signaling specificity and functional synergism of CBL-CIPK complexes, forming a truly complex CBL-CIPK network when plants confronting a variety of external changes such as nutrient ions deprivation and abiotic stresses (Luan, 2009).

Soil salinization is one of the major environmental stress that reduces plant growth and productivity throughout the world, affecting an estimated 45 million hectares of irrigated land (Huang et al., 2019a). Under salt stress, extracellular high salt environment increased intracellular osmotic pressure and accumulated intracellular Na⁺ to a toxic level (Cheeseman, 1988; Deinlein et al., 2014). Although osmotic pressure and Na⁺ stress sensors in plants have not yet been identified, it seems that such stress signal transduction is closely related to Ca²⁺ pathway (Cheeseman, 1988). The SOS (salt overly sensitive) pathway was originally discovered from the model plant Arabidopsis and was a well-studied Ca²⁺-dependent CBL-CIPK module involved in salinity stress response regulation in plants (Olías et al., 2009). The SOS pathway, which involves two Ca²⁺ sensor proteins, SOS3/AtCBL4 and SCaBP8/AtCBL10; the protein kinase SOS2/AtCIPK24; and the PM (plasma membrane)- Na⁺/H⁺ antiporter SOS1/AtNHX7 (Liu et al., 2020). The Ca²⁺ signal induced by high-salt is decoded by SOS3 and SCaBP8. SOS3 recruit SOS2 to the PM and activate its kinase activity. SOS2/SOS3 complex then phosphorylates and activates downstream PM-binding AtNHX7/SOS1 to transport Na⁺ out of the cell (Ji et al., 2013). Besides, AtCIPK24 can interact with AtCBL10, then phosphorylates and activates the vacuolar NHX, pumping excess Na⁺ into the vacuole. The SOS pathway also exists in many other plants, such as rice, poplar (Tang et al., 2010), and spinach (Zhao et al., 2020). In rice, OsCIPK24/OsSOS2 interacts with OsCBL4/OsSOS3 regulating the activity of PM-located OsSOS1, thus participating in the regulation of plant salt tolerance (Pandey et al., 2015).

Lonicera japonica belongs to the honeysuckle family and is widely cultivated as an ornamental plant. Its dried flower buds have been prescribed in traditional Chinese medicine (TCM) to treat fever, influenza, sores and swelling for thousands of years (Pu et al., 2020). It is also a crucial antiviral drug used to treat the SARS coronavirus, influenza A viruses, the H1N1, H5N1, H7N9 flu virus, and Enterovirus 71 in recent years (Liu et al., 2005; Shang et al., 2011). It has further value as a component of cosmetic, healthy food, and beverages due to its unique aroma and pharmacological activity. Previously, Huang et al. found that the honeysuckle cultivar “Huajin 6” has high salt tolerance, but the physiological and molecular mechanisms are remained elusive until now (Huang et al., 2019b). Due to the great significance of CBL-CIPK networks in various physiological and stress resistance processes, it is of great significance to explore the salt tolerance mechanism of honeysuckle start from the analysis of CBL-CIPK gene.

In this study, we identified 6 CBL and 17 CIPK genes based on the genome sequences of honeysuckle. Various characteristics of these genes were analyzed, including physicochemical properties, phylogenetic relationship, conserved motifs, gene structures, cis-acting elements analysis, protein three-dimensional (3-D) structure prediction, and putative protein-protein interaction (PPI) prediction. Additionally, the gene expression patterns of CBL-CIPK genes were analyzed in different tissues and gradient salt stress. Our results could reveal the roles of CBL-CIPK genes in response to high-salt environments, and our study aimed to provide a valuable reference for further utilization of CBL-CIPK genes to develop salt-tolerant medicinal plants in the context of the global reduction of cultivated land.

Honeysuckle plays an irreplaceable role in the development of TCM. The completion of the whole-genome sequencing of honeysuckle (unpublished) makes it possible to analyze various gene families of honeysuckle by bioinformatics. Here, a total of six LjCBL genes and 17 LjCIPK genes have been identified and characterized. There are 10 LjCBL genes and 26 LjCIPK genes in Arabidopsis (Arabidopsis thaliana), 10 OsCBL genes and 33 OsCIPK genes in rice (Oryza sativa) (Kolukisaoglu et al., 2004; Kanwar et al., 2014), seven VvCBL genes and 20 VvCIPK genes in Grapevine (Vitis vinifera) (Xi et al., 2017), 10 PtCBL genes and 27 PtCIPK genes in poplar (Populus trichocarpa) (Tang et al., 2010), five SmCBL genes and 15 SmCIPK genes in eggplant (Solanum melongena) (Li et al., 2016), eight ZmCBLs and 43 ZmCIPK in maize (Zea mays) (Chen et al., 2011), 19 BrrCBL and 51 BrrCIPK genes in Turnip (Brassica rapa) (Yin et al., 2017). It is well known that gene duplication and loss events during evolution lead to these differences in the number of CBL-CIPK genes among various species (Tian et al., 2017). The presence of multiple CBL-CIPK genes in the genome of the honeysuckle indicates the functional diversity of the CBL-CIPK gene family.

CBL proteins share an overall structural homology consisting of four elongation factor EF-hand domains responsible for binding Ca²⁺. Differences in the EF-hand motifs may result in different ways of binding to Ca²⁺ (Raymond, 1998; Kim et al., 2000; Tang et al., 2020). As shown in Table 1, EF-hands of LjCBLs were highly conserved in honeysuckle, indicating the functional conservation of binding Ca²⁺ ions. CIPK consists of a conserved N terminal catalytic domain for kinase activity and a less conserved C terminal regulatory region containing the NAF motif/FISL motif for interaction with CBL as well as the protein phosphatase interaction motif for interaction with type-2C protein phosphatases (PP2C) and is separated by a junction domain (Albrecht et al., 2001; Liu et al., 2019). Once signal transduction begins, Ca²⁺-bound CBL interacts with CIPK via the NAF domain and releases the inhibitory effect of the C terminus, leading to activation of the kinase (Guo et al., 2001; Tang et al., 2020). It has been shown that the putative NAF domains are highly conserved in the CIPK gene families, which is specific to mediate the interaction between CBL and CIPK (Zhang et al., 2019). Motif analysis showed that the NAF domain was located in motif 10 of the LjCIPKs and it existed in all the detected CIPKs in honeysuckle. Similar results were found in pepper (Capsicum annuum) (Ma X. et al., 2019) and canola (Brassica napus) (Zhang et al., 2014). These results indicated the CIPK family genes in honeysuckle are relatively conserved during evolution.

Protein myristoylation and palmitoylation are two critical modifications necessary for protein stability, aggregation, and trafficking (Kim et al., 2000; Hemsley and Grierson, 2008). Myristoylation is an irreversible protein modification in which myristate, a 14-carbon saturated fatty acid, is covalently attached through an amide bond to an N-terminal glycine residue in a co-translational process (Ishitani et al., 2000). In honeysuckle, LjCBL1/2/4/6 starts from a conserved N-myristoylation site, similar to the CBL protein structures from canola (Brassica napus) (Zhang et al., 2014) and rice (Oryza sativa) (Gu et al., 2008), which may play a role in the membrane targeting of the CBL-CIPK complex. Palmitoylation is the reversible addition of fatty acids to proteins, which increases their membrane affinity (Hemsley and Grierson, 2008). In Arabidopsis, the S-acylation-dependent (S-acylation, also known as palmitoylation) association of AtCBL2 with the vacuolar membrane is essential for ABA responses (Hrabak et al., 2003). Consistent with the results of Arabidopsis and canola (Brassica napus) (Zhang et al., 2014), all the six LjCBL proteins possess typical palmitoylation sites at the N-terminal.

The phylogenetic analysis serves as an excellent method to analyze evolutionary relationships among genes. Phylogenetic analysis of LjCBL and LjCIPK genes in honeysuckle, together with Arabidopsis and rice, classified both CBLs and CIPKs mainly into four different groups. Similar results have also been found in eggplant (Solanum melongena) (Li et al., 2016) and canola (Brassica napus) (Zhang et al., 2014). The results also demonstrated that the CBL-CIPK were unevenly distributed among subfamilies, and the CBL-CIPK gene family members in honeysuckle, rice, and Arabidopsis subfamilies. Consistent with the current information of plant evolution, the phylogenetic tree of LjCBLs and LjCIPKs were more closely related to Arabidopsis (dicot) compared to rice (monocot). However, several CBL-CIPK presented in Arabidopsis but was absent in honeysuckle, which mainly because of gene loss. The evolutionary characteristics of exon/intron structure provide strong evidence for phylogenetic grouping (Boudet, 2001). For LjCBLs, all of the CDS was discontinuous by the presence of introns. Whereas, CIPK genes were divided into an exon-poor clade (Group i/ii/iii) and an exon-rich clade (Group iv), similar to those in Arabidopsis (Mao et al., 2016), pepper (Ma X. et al., 2019), and cotton (Cui Y. et al., 2020). Combined with the evolutionary analysis of CIPK in plants, our results indicate that intron gain or loss events were the major driving factors for the gene structural evolution of the CIPK gene family before eudicot-monocot divergence (Zhu et al., 2016; Cui Y. et al., 2020). The structural diversification among the CBL-CIPK genes families may allow CBL-CIPK genes to function differently (Yin et al., 2017).

Gene duplication provides opportunities for the new gene production and its functional divergence in the process of gene family expansion and evolution. The paralogous genes were generated during the divergent evolution from a common ancestral gene through duplication events (segmental or tandem duplication) (Xi et al., 2017). It has been shown that the expansion of the CIPK gene family in Gossypium hirsutum and Gossypium barbadense mainly due to whole-genome duplication and segmental duplications (Cui Y. et al., 2020). In honeysuckle, only one CIPK paralogous pair (LjCIPK10/LjCIPK15) was identified and were generated by segmental duplication events, as the genes present on different chromosomes. The Ka/Ks ratio is used to identify whether selective pressure existed on amino acid substitutions (Nekrutenko et al., 2002). Our results suggested that the function of the duplicated CIPK genes in honeysuckle did not diverge much during their evolution course, and purifying selection could mainly contribute to the maintenance of function in CIPK gene families. Noticeably, the duplicated genes of LjCIPKs were only detected in the intron-poor clade, a similar phenomenon has been observed in Grapevine (Vitis vinifera), indicating that the intron-poor clade of the LjCIPK gene family may play a more specific role to fulfill the specific characteristics of honeysuckle (Xi et al., 2017).

As a key molecular switch, cis-acting regulatory elements participate in the transcriptional regulation of gene activities that control various biological processes (Wu et al., 2019). Hormones, such as ABA, salicylic acid (SA), auxin (IAA), and gibberellin, play critical roles in several developmental stages and stress response (Gallego-Giraldo et al., 2008; Mishra et al., 2014; Li et al., 2019; Zhang and Li, 2019). Here, seven regulatory elements related to hormones were identified in the promoters of CBL-CIPK genes. ABRE (ABA-responsive element), which belongs to the so-called G-box family, contains an ACGT core, a sequence known to be recognized by plant bZIP proteins. ABRE was found in two-thirds of LjCBL and 13 out of 17 LjCIPK genes, respectively, which was also identified in both Arabidopsis and rice (Gomez-Porras et al., 2007), suggesting that the most CBL-CIPK genes might be involved in the ABA signal pathway, which mainly controls stomatal closure, seed and bud dormancy, and physiological responses to cold, drought, and salinity stress (Mishra et al., 2014). Furthermore, seven stress-responsive regulatory elements were identified namely ARE, W box, LTR, STRE, MBS, TC-rich repeats. Similar elements were found in CBL-CIPKs from Grapevine (Vitis vinifera) and BnaNHXs from oilseed rape (Brassica napus) (Cui J.-q. et al., 2020). Interestingly, W box (TTGACC) was identified in half of the CBL-CIPK genes from honeysuckle. W box is recognized by the family of WRKY transcription factors, which is involved in certain developmental processes and stress response, such as drought stress response in tomato (Ahammed et al., 2020), salt response in Arabidopsis (Xu et al., 2018) and populus (Jiang et al., 2020). In general, the identified cis-acting elements here help in understanding their roles in the developmental and various biotic stress-related mechanisms.

All CBL-CIPK gene expression displayed tissue specificity, which indicated the distinct effect of CBL-CIPK genes. Interestingly, LjCBL3 and LjCIPK3/9/10/14/15 were highly expressed in mature leaves. Same result was found in cassava (Manihot esculenta), MeCBL1/9 and MeCIPK23 have higher transcriptional levels in the mature stage (Mo et al., 2018). Since CBL-CIPK is a modulator system for efficient nutrient acquisition, it is speculated that different tissues have different requirements for nutrient ions, resulting in tissue-specific expression of related genes (Dong et al., 2021; Verma et al., 2021). CBL and CIPK genes have been reported to enhance salinity tolerance in different species by establishing the homeostasis of macro-nutrients in the cytosol and subcellular compartments, such as in pigeon pea (Cajanus cajan) (Meng et al., 2021) and cotton (Gossypium hirsutum) (Sun et al., 2021). Our study revealed that in honeysuckle, several CBL-CIPK genes express differentially at different time intervals under salinity stress. The SOS pathways, consisting of SOS3, SOS2, and SOS1, have been well defined as crucial pathways to control cellular ion homeostasis, by extruding Na⁺ to the extracellular space, thus conferring salt stress resistance in plants. As shown in Table 1, LjCBL4 exhibited orthologous relationships with AtSOS3(At5g24270), while LjCIPK16 exhibited orthologous relationships with AtSOS2(At5g35410). As shown in Figure 1, there is a close evolutionary relationship between LjCBL4 and SOS3, LjCIPK16 and SOS2. Interestingly, SOS1, a plasma membrane Na⁺/H⁺ antiporter, was predicted to interact with LjCIPK15/16 and LjCBL4 in the PPI network, while NHX1, the Na⁺/H⁺ antiporter located on tonoplast, was also predicted to interact with LjCIPK16 and LjCBL4, which indicated the key role of LjCIPK16 and LjCBL4 proteins in the salt tolerance of honeysuckle. Moreover, LjCBL4, LjCIPK16 and SOS1 were clustered into green cluster in the PPI network, indicating that they may act in the same pathway. As shown in Figure 6, expression profile analyses revealed that LjCBL4 and LjCIPK16 were upregulated under salt stress. The SOS pathway genes upregulated in response to salinity stress were confirmed in many plants, such as spinach (Spinacia oleracea) (Zhao et al., 2020) and poplar (Populus trichocarpa) (Tang et al., 2010). This result suggested that the Ca²⁺-SOS3-SOS2-cation/H⁺ antiporters (NHXs/AKTs) pathway might be not only a common but also an essential pathway regulating plant salt stress resistance (Zhao et al., 2020). This result also demonstrated the conservation of SOS pathway genes in honeysuckle and a model of Ca²⁺-LjCBL4/LjSOS3-LjCIPK16/LjSOS3 module-mediated abiotic stress signaling in honeysuckle is proposed.

At the same time, the functions of LjCBL2/5, LjCIPK1/11/13/15/17 in the response of honeysuckle to salt stress are worth further exploring. Notably, LjCIPK1 and LjCIPK15 show greater enhanced expression under salt stress, even higher than LjCIPK16. LjCIPK15 exhibited orthologous relationships with AtCIPK6 (At4g30960). In Arabidopsis, overexpression of AtCIPK6 increased plant tolerance to salt stress (Chen et al., 2013); cipk6 mutant was more sensitive to salt stress compared to wild-type (Tripathi et al., 2009), which indicated LjCIPK15 may play significant roles in salt tolerance. LjCIPK1 exhibited orthologous relationships with AtCIPK11 (At2g30360). It has been shown that AtCIPK11 functions as a negative regulator in drought stress response in Arabidopsis, its role in salt tolerance needs deep exploration (Ma Y. et al., 2019). Although the sensitivity of each gene to external environmental changes is different, their role in the response of honeysuckle to salt stress deserves more attention.

In the present study, a total of six LjCBL and 17 LjCIPK genes were identified. The phylogenetic analysis divided both CBL and CIPK genes into four subgroups and the same clade had similar motif compositions and gene structures. Cis-Acting elements analysis implied that both LjCBLs and LjCIPKs are involved in stress adaptation. PPI network analysis results showed that LjCBL4 is hypothesized to interact with LjCIPK7/9/15/16 and SOS1/NHX1. The salt-induced expression patterns confirmed that the expression levels of LjCBL2/4, LjCIPK1/15/16/17 were affected by salinity. The theoretical foundation was established in the present study for the further functional characterization of the CBL-CIPK gene families in honeysuckle. However, extra works are required to decipher the interaction networks between LjCBLs and LjCIPKs, and the regulation mode of CBL-CIPK complexe’s response to salt stress.