Genome-Wide Identification and Expression Profiling of CBL-CIPK Gene Family in Pineapple (Ananas comosus) and the Role of AcCBL1 in Abiotic and Biotic Stress Response

Ca2+ serves as a ubiquitous second messenger regulating several aspects of plant growth and development. A group of unique calcium sensor proteins, calcineurin B-like (CBL), interact with CBL-interacting protein kinases (CIPKs) to decode the Ca2+ signature inside the cell. Although CBL-CIPK signaling toolkit has been shown to play significant roles in the responses to numerous stresses in different plants, the information about pineapple CBL-CIPK remains obscure. In the present study, a total of eight AcCBL and 21 AcCIPK genes were identified genome-wide in pineapple. The identified genes were renamed on the basis of gene ID in ascending order and phylogenetic analysis divided into five groups. Transcriptomic data analysis showed that AcCBL and AcCIPK genes were expressed differentially in different tissues. Further, the expression analysis of AcCBL1 in different tissues showed significant changes under various abiotic stimuli. Additionally, the ectopic expression of AcCBL1 in Arabidopsis resulted in enhanced tolerance to salinity, osmotic, and fungal stress. The present study revealed the crucial contribution of the CBL-CIPK gene in various biological and physiological processes in pineapple.


Introduction
In living organisms, the precise perception and timely decoding of environmental and developmental signals are essential for survival. The signal transduction pathway involved in this process includes several indispensable components including calcium (Ca 2+ ), which serves as a ubiquitous second messenger in all eukaryotes [1][2][3]. Various organelles such as endoplasmic reticulum (ER), mitochondria, and vacuoles work as Ca 2+ store and help in the maintenance of a critical balance of Ca 2+ inside the cell [4]. Acknowledging the stress or developmental signals, the cytosolic calcium concentration rapidly shoots up as an early response [5]. Consistently, in plants, the rapid increase of cytosolic Ca 2+ concentration is well documented against several stress factors such as salinity, genes and performed a comprehensive analysis including exon-intron organization, chromosome distribution, and phylogenetic analysis. Additionally, we have performed expression analysis to identify the involvement of these genes in different biological and developmental stages in pineapple. We further characterized AcCBL1 by overexpressing it in Arabidopsis. Our study provides a basis for further utilization of CBL-CIPK genes to develop next-generation crops especially in the context of a globally changing environment.

Identification of CBL-CIPK Genes in Pineapple Genome
The CBL and CIPK gene sequences from Arabidopsis and Oryza sativa were downloaded from TAIR (http://www.arabidopsis.org) and China Rice Data Center (http://www.ricedata.cn/gene/index.htm) respectively. Further, the CBL and CIPK genes were used as keywords against the pineapple genome database of phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) to identify CBL-CIPK genes from pineapple. Additionally, Hidden Markov Model (HMM) profile of the NAF domain (PF03822) from the Pfam database (http://pfam.xfam.org/) and EF-hand calcium-binding domain (PS50222) from prosite (https://prosite.expasy.org/) were downloaded. We then performed searches from the pineapple genome using the HMM profiles by BLAST-P with the e-value set at 0.01. The identified sequences were further verified and redundant sequences were removed from the list. Finally, ExPASy (http://web.expasy.org/compute_pi/) was used determine the isoelectric point (pI) and molecular weight (MW) of CBL and CIPK proteins of pineapple.

Phylogenetic Analysis
The phylogenetic relationship of CBL-CIPK proteins between pineapple, Arabidopsis, and rice were studied using identified CBL-CIPK amino acid sequences from pineapple, Arabidopsis, and rice. The multiple sequence alignments were performed by using MUSCLE 3.7 and the phylogenetic trees were constructed by MEGA7 using the Maximum likelihood (ML) method with default parameters with the bootstrap option n = 1000.

Chromosome Location of CBL and CIPK Genes in Pineapple
The information about the location of AcCBL and AcCIPK genes on chromosomes were collected from phytozome. MapChart software was used to visualize the AcCBL and AcCIPK genes mapped on chromosomes based on the gene start position and the length of related chromosomes.

Pineapple Growth and Treatments
Two-month-old tissue culture raised pineapple (Ananas comosus) variety MD2 plants were obtained from Qin lab (http://www.qinlab.net) [44] and grown in plastic pots containing soil mix (peat moss:perlite = 2:1 (v/v)) placed at 30 • C with 16 h light/8 h dark photoperiod under an intensity of 70 µmol m −2 s −1 70% humidity as described earlier by Chen et al. [45]. For the various stress treatment experiments, the pineapple plants were exposed to different treatments for 24,48, and 72 h. The treatments included salinity stress (150 mM NaCl), osmotic stress (350 mM mannitol), heat (45 • C), and cold stress (4 • C) respectively. The leaf and root tissues from whole plants were harvested and frozen immediately in liquid nitrogen and stored at −80 • C until used.

Arabidopsis Treatments and Root Growth Assay
The wild-type Arabidopsis thaliana (L.) Heynh (Col-0; CS60000) was obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). All plants used were Columbian in background. Surface sterilized Arabidopsis seeds were placed in a round, 9 cm Petri plates on modified Hoagland's medium [46,47] containing 1% (w/v) sucrose and 1% (w/v) agar. The plates were kept at 4 • C in the dark for 2 day for seed stratification. After stratification, the plates were transferred to a growth room at 22 • C with 16 h light/8 h dark photoperiod under an intensity of 100 µmol m −2 sec −1 and seedlings were grown vertically.
Salinity stress treatment and growth recovery were performed from T3 generation of 3 different AcCBL1 overexpressing lines. 5-day-old seedlings were transferred to new plates containing the modified Hoagland medium supplemented with 150 mM NaCl and kept 10 days in a growth room. Primary roots were analyzed after 2 days, 5 days, and 10 days of transfer. The experiments were repeated at least three times with 8-10 seedlings per treatment. To measure salinity induced root growth inhibition seedlings were photographed by digital camera (NIKON D750, Japan) and root growth was analyzed by ImageJ software (http://rsbweb.nih.gov/ij/).
For in-situ detection of hydrogen peroxide induced against biotic stress after exposure to fungus (Sclerotinia sclerotiorum) mature Arabidopsis rosette leaves were exposed to fungus for 24 h followed by staining with 3,3-diaminobenzidine (DAB) as reported earlier by Daudi et al. [48].

RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA from desired tissues was isolated by using the RNeasy kit (Qiagen, MD, USA), followed by treatment with DNase I (Thermo Fisher Scientific, CA, USA) and first-strand cDNA synthesis by reverse transcription using the ThermoScript RT-PCR kit (Thermo Fisher Scientific, CA, USA). Quantitative PCR was performed by using FastStart DNA Master SYBR Green I master mix (Takara, Shiga, Japan) in a CFX96 qPCR system (Bio-Rad, Singapore). Primers used in qRT-PCR are listed in supplementary Table S3. The PCR cycle conditions for quantitative PCR were as follows: 95 • C for 2 min followed by 40 cycles of 95 • C for 10 s, 60 • C for 15 s, and 72 • C for 15 s. For each analysis, two technical replicates from three biological replicates were performed and pineapple EF1a was used as an internal control to normalize the mRNA levels, and finally, the fold change of genes was calculated using the 2 −∆∆CT method.

RNA-Seq and Data Analysis
RNA was extracted from different developmental stages as described earlier by Chen et al., [45], pineapple of MD2 variety through Plant RNeasy Mini Kit (Qiagen MD, USA). Illumina sequencing was performed as previously described [45], with 1 µg RNA per sample and two independent biological replicates per genotype. cDNA libraries were constructed using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (NEB, MA, USA) following standard protocols. The transcript abundance of pineapple CBL-CIPK genes were calculated as fragments per kilobase of exon model per million mapped reads (FPKM). The heatmap was created by pheatmap packages in R based the log2 (FPKM + 1).

Vector Constructs
AcCBL1:GFP was generated by amplifying 678 bp of coding sequence without the stop codon from pineapple cDNA using the primers listed in Table S3. The amplified PCR fragment was then cloned into the pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA). pENTR/D-TOPO clones were then recombined into the destination vector pGWB505 by using LR Clonase II (Invitrogen, Carlsbad, CA, USA). Before performing the bacterial and plant transformation, the construct was confirmed by sequencing.

Live Cell Imaging
For live cell microscopy, five-day-old AcCBL1 overexpressing plants were treated either by 150 mM salt or wounded manually. After the treatment, the seedlings were mounted in the water on a cover glass for observation on a TCS SP8 microscope (Leica, Wetzlar, Germany) and imaged.

Statistical Analysis
Results are expressed as the means ± standard error (SE) from at least 3 experiments. To analyze statistical significance, a two-tailed Student's t-test was used.

Identification of the CBL-CIPK Genes in Pineapple
A total of 8 CBL and 26 CIPK genes were initially obtained from the pineapple genome using BLAST, HMM search, and available pineapple annotation. Five redundant CIPK genes were removed after the Pfam NAF domain (PF03822) investigation identified putative CIPK genes. Finally, a total of 8 CBLs and 21 CIPK gene models were selected and annotated based on their gene ID in ascending order (Table 1a,b). All the CBLs contained the EF-hand and CIPKs possess the conserved NAF/FISL motif. The detailed information of the identified CBL-CIPK genes such as gene name, gene ID, protein length, isoelectric points, and molecular weights is listed in Table 1. Among 8 identified CBLs, AcCBL6 was the smallest having 193 amino acid and AcCBL4 was largest with 359 amino acid, whereas AcCIPK8 was smallest with 382 amino acids and AcCIPK20 with 506 amino acid was largest in the group. The molecular weight of the AcCBL proteins ranged from 21.9 kDa to 40.8 kDa and 43.7 kDa to 55.8 kDa for CIPK proteins.
The evolutionary relationships and functional associations of CBL-CIPK genes in pineapple were investigated by constructing a multi-species phylogenetic tree using full-length protein sequences of CBL ( Figure 2) and CIPK ( Figure 3) from pineapple, Arabidopsis, and rice using the Maximum Likelihood method. AcCBLs in phylogenetic trees formed four subfamilies more closely related to rice compared to Arabidopsis. The tree also divided the AcCBLs into 5 subfamilies viz. AcCBL1 and AcCBL7 formed a group with OsaCBL2 and OsaCBL3, AcCBL5 and AcCBL6 formed a group with OsaCBL1, and AcCBL6 paired with OsaCBL6 ( Figure 2). Interestingly, some groups (depicted without any color in Figures 2 and 3) both among CBL and CIPK did not have any members from pineapple, indicating the evolutionary divergence between monocotyledonous and dicotyledonous plants species.  The evolutionary relationships and functional associations of CBL-CIPK genes in pineapple were investigated by constructing a multi-species phylogenetic tree using full-length protein sequences of CBL ( Figure 2) and CIPK ( Figure 3) from pineapple, Arabidopsis, and rice using the Maximum Likelihood method. AcCBLs in phylogenetic trees formed four subfamilies more closely related to rice compared to Arabidopsis. The tree also divided the AcCBLs into 5 subfamilies viz. AcCBL1 and AcCBL7 formed a group with OsaCBL2 and OsaCBL3, AcCBL5 and AcCBL6 formed a group with OsaCBL1, and AcCBL6 paired with OsaCBL6 ( Figure 2). Interestingly, some groups (depicted without any color in Figures 2 and 3) both among CBL and CIPK did not have any members from pineapple, indicating the evolutionary divergence between monocotyledonous and dicotyledonous plants species.

Chromosomal Distribution of Pineapple CBL-CIPK Genes
The pineapple CBL-CIPK genes were unevenly distributed on 15 linkage groups (LG) out of 25 linkage groups present in the genome. The linkage groups LG 06, 09, 14,15,17,21,22,23,24, and 25 did not contain any CBL or CIPK genes ( Figure 4). All 8 CBLs were located on different linkage groups, whereas CIPKs were located on different linkage groups ranging from 1-4 in number. Linkage groups LG01, 03, 07, 12, 16, and 19 had a single CIPK gene, LG 02, 08, 13, and 18 had two CIPK genes, LG05 had 3 CIPK genes and maximum 4 CIPK genes were located on LG 04 ( Figure 4). The result showed no correlation between the length of linkage groups and number of CBL-CIPK gene distribution.

Chromosomal Distribution of Pineapple CBL-CIPK Genes
The pineapple CBL-CIPK genes were unevenly distributed on 15 linkage groups (LG) out of 25 linkage groups present in the genome. The linkage groups LG 06, 09, 14,15,17,21,22,23,24, and 25 did not contain any CBL or CIPK genes ( Figure 4). All 8 CBLs were located on different linkage groups, whereas CIPKs were located on different linkage groups ranging from 1-4 in number. Linkage groups LG01, 03, 07, 12, 16, and 19 had a single CIPK gene, LG 02, 08, 13, and 18 had two CIPK genes, LG05 had 3 CIPK genes and maximum 4 CIPK genes were located on LG 04 ( Figure 4). The result showed no correlation between the length of linkage groups and number of CBL-CIPK gene distribution.

Expression Profiling of Pineapple CBL-CIPK Genes in Different Developmental Stages
The expression profiling of all the CBL-CIPK genes was carried out from RNA-Seq data generated from different organs of the pineapple plant. The expression profiles of all the pineapple CBL-CIPK genes were generated and hierarchically clustered using average log values of each gene ( Figure 5). The hierarchical clustering of RNA-Seq data divided them into three major groups. In group I, low expressed genes (AcCIPK4, AcCIPK5, AcCIPK9, and AcCIPK11) were clustered together. In contrast, group II contained genes (6 AcCBLs and 13 AcCIPK genes) with high level of expression in all the selected stages ( Figure 5). Whereas, group III had genes (2 AcCBLs and 4 AcCIPK genes) with relatively low levels of expression. The expression pattern of CBL-CIPK genes suggests that they are expressed differentially during different developmental stages and contribute essentially to plant growth and development.

Expression Profiling of Pineapple CBL-CIPK Genes in Different Developmental Stages
The expression profiling of all the CBL-CIPK genes was carried out from RNA-Seq data generated from different organs of the pineapple plant. The expression profiles of all the pineapple CBL-CIPK genes were generated and hierarchically clustered using average log values of each gene ( Figure 5). The hierarchical clustering of RNA-Seq data divided them into three major groups. In group I, low expressed genes (AcCIPK4, AcCIPK5, AcCIPK9, and AcCIPK11) were clustered together. In contrast, group II contained genes (6 AcCBLs and 13 AcCIPK genes) with high level of expression in all the selected stages ( Figure 5). Whereas, group III had genes (2 AcCBLs and 4 AcCIPK genes) with relatively low levels of expression. The expression pattern of CBL-CIPK genes suggests that they are expressed differentially during different developmental stages and contribute essentially to plant growth and development.

Expression Profiling of Pineapple CBL1 Gene in Response to Different Treatments
In the RNA-Seq experiment AcCBL1 gene showed the highest expression, so we selected it for further study and investigated the role of AcCBL1 gene during abiotic stress response. For this, we used quantitative real-time PCR (qRT-PCR) and determined the spatial and temporal expression profile of AcCBL1 in different tissues (root and leaf) of pineapple at different time intervals (0, 24, 48, and 72 h) after salinity stress (150 mM NaCl), osmotic stress (350 mM mannitol), heat (45 °C), and cold stress (4 °C) treatments ( Figure 6). The qPCR data showed that compared to untreated plants the transcript level of AcCBL1 significantly increased during salinity, osmotic, and heat treatments whereas, the cold stress did not show significant response. The results also showed the differential expression of AcCBL1 when it was compared between root and leaf ( Figure 6). Collectively, these results strongly suggest that the AcCBL1 gene contributes to various abiotic stresses in pineapple.

Expression Profiling of Pineapple CBL1 Gene in Response to Different Treatments
In the RNA-Seq experiment AcCBL1 gene showed the highest expression, so we selected it for further study and investigated the role of AcCBL1 gene during abiotic stress response. For this, we used quantitative real-time PCR (qRT-PCR) and determined the spatial and temporal expression profile of AcCBL1 in different tissues (root and leaf) of pineapple at different time intervals (0, 24, 48, and 72 h) after salinity stress (150 mM NaCl), osmotic stress (350 mM mannitol), heat (45 • C), and cold stress (4 • C) treatments ( Figure 6). The qPCR data showed that compared to untreated plants the transcript level of AcCBL1 significantly increased during salinity, osmotic, and heat treatments whereas, the cold stress did not show significant response. The results also showed the differential expression of AcCBL1 when it was compared between root and leaf ( Figure 6). Collectively, these results strongly suggest that the AcCBL1 gene contributes to various abiotic stresses in pineapple.

Salt Stress Increases the Localization of AcCBL1 in Arabidopsis Roots
Having established the role of AcCBL1 in salinity, osmotic, and heat stress in pineapple. We further investigated the biological function of AcCBL1 by ectopically expressing AcCBL1 gene in Arabidopsis under CaMV 35S promoter and GFP tag. Using a confocal microscope we monitored the localization of AcCBL1:GFP after transfer experiment. Transfer experiment was carried out with five-day-old seedlings by transferring them to a fresh plate consisting of Hoagland medium supplemented with 150 mM NaCl for 48 h. The results showed that the AcCBL1:GFP mainly localized in the cytosol and its expression increased after salt stress treatment (Figures 7 and 8b). The increased localization of AcCBL1 in Arabidopsis roots clearly indicates its involvement in salt stress. Taken together, our results indicate that the AcCBL1 helps the plant to thrive well under salt stress.

Injury Induces AcCBL1 Translocation From Cytosol to the Nucleus
Previous studies have established that Arabidopsis CBLs are induced during wounding and injury [50]. In order to investigate the possible role of AcCBL1 during injury, we monitored the expression of AcCBL1:GFP after manually injuring the AcCBL1 overexpressing plants. Interestingly, we found that the expression of AcCBL1 was regulated by wounding and AcCBL1 was translocated to the nucleus after the injury (Figure 8a). The results clearly indicate that the AcCBL1 is regulated by injury and may be involved in the protection against injury stress.

Salt Stress Increases the Localization of AcCBL1 in Arabidopsis Roots
Having established the role of AcCBL1 in salinity, osmotic, and heat stress in pineapple. We further investigated the biological function of AcCBL1 by ectopically expressing AcCBL1 gene in Arabidopsis under CaMV 35S promoter and GFP tag. Using a confocal microscope we monitored the localization of AcCBL1:GFP after transfer experiment. Transfer experiment was carried out with five-day-old seedlings by transferring them to a fresh plate consisting of Hoagland medium supplemented with 150 mM NaCl for 48 h. The results showed that the AcCBL1:GFP mainly localized in the cytosol and its expression increased after salt stress treatment (Figures 7 and 8b). The increased localization of AcCBL1 in Arabidopsis roots clearly indicates its involvement in salt stress. Taken together, our results indicate that the AcCBL1 helps the plant to thrive well under salt stress.

Injury Induces AcCBL1 Translocation from Cytosol to the Nucleus
Previous studies have established that Arabidopsis CBLs are induced during wounding and injury [50]. In order to investigate the possible role of AcCBL1 during injury, we monitored the expression of AcCBL1:GFP after manually injuring the AcCBL1 overexpressing plants. Interestingly, we found that the expression of AcCBL1 was regulated by wounding and AcCBL1 was translocated to the nucleus after the injury (Figure 8a). The results clearly indicate that the AcCBL1 is regulated by injury and may be involved in the protection against injury stress.

Overexpression of AcCBL1 Results in Resistance to Salinity, Osmotic, and Biotic Stress
As shown in Figure 6 and Figure 7, the expression of AcCBL1 is significantly increased during osmotic and salinity stress, suggesting that this gene may be participating in abiotic stress responses including salinity stress. Previously, the involvement of AtCBL1 and AtCBL10 in response to salt stress has been well documented [17,35,51]. To test whether AcCBL1 contributes to salinity stress we took different approaches: 1) We performed seedling transfer experiment of AcCBL1 overexpressing lines and observed the salinity effect on primary root growth and percent root growth compared to wild-type plants; 2) tested the germination and growth phenotype under continuous salt and osmotic stress; 3) checked the performance of AcCBL1 overexpressing lines under saline irrigation and 4) performed the fresh weight analysis of seedling after 10 days (d) of transfer experiment. In agreement to our hypothesis, AcCBL1 overexpressing Arabidopsis lines showed better growth performance on 150 mM NaCl containing plates when compared to wild-type plants in terms of percent root growth (Figure 9a), and primary root growth (Figure 9b,c), after 2 d and 5 d of the transfer. The AcCBL1 overexpressing lines also showed better germination rates under salt and osmotic (300 mM mannitol) stress (Figure 9d). Consistently, fresh weights of the seedling grown on medium containing NaCl were significantly more compared to wild type plants (Figure 9e,g). Moreover, the saline irrigation of 15 d old plants with 250 mM NaCl for 10 d also showed better

Overexpression of AcCBL1 Results in Resistance to Salinity, Osmotic, and Biotic Stress
As shown in Figures 6 and 7, the expression of AcCBL1 is significantly increased during osmotic and salinity stress, suggesting that this gene may be participating in abiotic stress responses including salinity stress. Previously, the involvement of AtCBL1 and AtCBL10 in response to salt stress has been well documented [17,35,51]. To test whether AcCBL1 contributes to salinity stress we took different approaches: (1) We performed seedling transfer experiment of AcCBL1 overexpressing lines and observed the salinity effect on primary root growth and percent root growth compared to wild-type plants; (2) tested the germination and growth phenotype under continuous salt and osmotic stress; (3) checked the performance of AcCBL1 overexpressing lines under saline irrigation and (4) performed the fresh weight analysis of seedling after 10 days (d) of transfer experiment. In agreement to our hypothesis, AcCBL1 overexpressing Arabidopsis lines showed better growth performance on 150 mM NaCl containing plates when compared to wild-type plants in terms of percent root growth (Figure 9a), and primary root growth (Figure 9b,c), after 2 d and 5 d of the transfer. The AcCBL1 overexpressing lines also showed better germination rates under salt and osmotic (300 mM mannitol) stress (Figure 9d). Consistently, fresh weights of the seedling grown on medium containing NaCl were significantly more compared to wild type plants (Figure 9e,g). Moreover, the saline irrigation of 15 d old plants with 250 mM NaCl for 10 d also showed better growth performance compared to wild-type (Figure 9f).
Additionally, to investigate the contribution of AcCBL1 overexpressing lines to resistance to biotic stresses, we infected the mature rosette leaves with plant pathogenic fungus (Sclerotinia sclerotiorum) for 24 h and performed DAB staining. The DAB staining results indicated that the AcCBL1 lines had less accumulation and distribution of hydrogen peroxide (Figure 10b), indicating the increased tolerance of these plants under biotic stress. Collectively, the above results convincingly show that the AcCBL1 contributes to abiotic and biotic stress and AcCBL1 overexpressing plants display better performance under unfavorable stress conditions. Biomolecules 2019, 9,293 14 of 20 growth performance compared to wild-type (Figure 9f). Additionally, to investigate the contribution of AcCBL1 overexpressing lines to resistance to biotic stresses, we infected the mature rosette leaves with plant pathogenic fungus (Sclerotinia sclerotiorum) for 24 h and performed DAB staining. The DAB staining results indicated that the AcCBL1 lines had less accumulation and distribution of hydrogen peroxide (Figure 10 b), indicating the increased tolerance of these plants under biotic stress. Collectively, the above results convincingly show that the AcCBL1 contributes to abiotic and biotic stress and AcCBL1 overexpressing plants display better performance under unfavorable stress conditions.

Discussion
Due to their sessile nature, plants face several biotic and abiotic stresses during their life cycle. Several genes contribute in defense and protective measure for plants to thrive well under hostile conditions [37,52,53]. CBL-CIPK gene family consists of plant-specific and calcium involving signaling modules indispensable to various stress signaling pathways. Due to availability of sequenced genome, CBL-CIPK gene families have been extensively studied in several plant species [19,[38][39][40][41][42]54] however, the information about pineapple CBL-CIPK genes remain elusive. In the present study, a comprehensive search of CBL-CIPK genes from pineapple genome resulted in the identification of eight CBL and 21 CIPK genes, which were designated as AcCBL1 to AcCBL8 and AcCIPK1 to AcCIPK21 on the basis of their gene ID in the order of increasing number.
In plants, the CBL-CIPK network represents an excellent module for decoding Ca 2+ signals during stress. This network is also a classic example of a diverged Ca 2+ signal decoding system for sensing differential Ca 2+ signals in numerous stress signaling pathways [42]. CBL proteins interact with different CIPK proteins depending on the cellular Ca 2+ signals (concentration) that determine the precise outcome due to specific complex formation [55]. A 24 amino acid domain, referred to as NAF domain (due to the presence of conserved Asn-Ala-Phe), is specific and sufficient to mediate the interaction between CBL and CIPK [18]. We also utilized NAF domain as a query to identify CIPK proteins in pineapple genome.
Gene structure (intron-exon structures) in the gene families are often considered as imprints of evolution [56]. During the genome duplication, the deletion and insertion or both could happen to the genes. The intron-exon structure of the identified AcCBL genes showed that the CDS of all the AcCBLs were discontinuous by the presence of introns (Figure 1a). Whereas, AcCIPK genes can be grouped in intron less or intron poor and intron rich genes (Figure 1b). The intron less or intron-poor and intron-rich genes of CIPKs are conserved among other genomes such as Arabidopsis, maize, grapevine, and Brassica [19,38,40,42]. The intron numbers of AcCBL and AcCIPK genes ranged from

Discussion
Due to their sessile nature, plants face several biotic and abiotic stresses during their life cycle. Several genes contribute in defense and protective measure for plants to thrive well under hostile conditions [37,52,53]. CBL-CIPK gene family consists of plant-specific and calcium involving signaling modules indispensable to various stress signaling pathways. Due to availability of sequenced genome, CBL-CIPK gene families have been extensively studied in several plant species [19,[38][39][40][41][42]54] however, the information about pineapple CBL-CIPK genes remain elusive. In the present study, a comprehensive search of CBL-CIPK genes from pineapple genome resulted in the identification of eight CBL and 21 CIPK genes, which were designated as AcCBL1 to AcCBL8 and AcCIPK1 to AcCIPK21 on the basis of their gene ID in the order of increasing number.
In plants, the CBL-CIPK network represents an excellent module for decoding Ca 2+ signals during stress. This network is also a classic example of a diverged Ca 2+ signal decoding system for sensing differential Ca 2+ signals in numerous stress signaling pathways [42]. CBL proteins interact with different CIPK proteins depending on the cellular Ca 2+ signals (concentration) that determine the precise outcome due to specific complex formation [55]. A 24 amino acid domain, referred to as NAF domain (due to the presence of conserved Asn-Ala-Phe), is specific and sufficient to mediate the interaction between CBL and CIPK [18]. We also utilized NAF domain as a query to identify CIPK proteins in pineapple genome.
Gene structure (intron-exon structures) in the gene families are often considered as imprints of evolution [56]. During the genome duplication, the deletion and insertion or both could happen to the genes. The intron-exon structure of the identified AcCBL genes showed that the CDS of all the AcCBLs were discontinuous by the presence of introns (Figure 1a). Whereas, AcCIPK genes can be grouped in intron less or intron poor and intron rich genes (Figure 1b). The intron less or intron-poor and intron-rich genes of CIPKs are conserved among other genomes such as Arabidopsis, maize, grapevine, and Brassica [19,38,40,42]. The intron numbers of AcCBL and AcCIPK genes ranged from 0 to 13 showing the large differences between the structures of the paralogs. This difference could be one of the attributes of functionality difference among the paralogs of CBL-CIPK genes. The phylogenetic analysis serves as an excellent method to analyze evolutionary relationships among genes [57]. In the present study, phylogenetic analysis of AcCBL and AcCIPK genes, together with Arabidopsis and rice, categorized AcCBLs mainly into five different groups ( Figure 2) and CIPKs also into five different groups (Figure 3). Consistent with the current information of plant evolution, in the phylogenetic tree AcCBLs were more closely related to rice CBLs (monocot) compared to Arabidopsis (dicot). These findings indicate that they may have arisen together by gene duplication via a common ancestor and could have similar functions. It is interesting to note that the lower plants contain a lesser number of CBL-CIPK genes; whereas their number remarkably increases in higher land plants. The abundance of CBL-CIPK genes could be due to the increase in the complexity of the land plants during their evolution [58]. We found that the pineapple genome contains eight CBL genes (Table 1a) compared to 10 CBLs of Arabidopsis and rice whereas, 21 CIPK genes (Table 1b)  Considering the importance of the CBL-CIPK genes in overall plant growth and response, we studied their expression profile in different tissues and development stages of pineapple. The expression profile suggests that AcCBL and AcCIPK genes express differentially in different tissues and stages ( Figure 5). The hierarchical clustering of the gene expression data grouped them into three different categories ( Figure 5). RNA-Seq data from different tissues and stages clearly indicated that the CBL-CIPK genes are involved in the regulation of different biological functions correlated to tissue or stage. For example, AcCIPK18 was highly expressed during the different stages of pineapple fruit development, suggesting that it may be involved in the regulation of pineapple fruit development ( Figure 5).
Increasing evidence suggests that the CBL-CIPK toolkit performs a plethora of functions in plant growth and development under optimal and stress conditions [6, [20][21][22]59]. In our study, we found that both the AcCBL1 and AcCBL7 were highly expressed in RNA-seq ( Figure 5). Therefore, we selected AcCBL1 to further investigate its expression under different stress conditions. Real-time quantitative RT-PCR analysis showed that the transcript level of AcCBL1 significantly changed during all the stress treatments applied. The AcCBL1 exhibited significant changes in root and leaf at all the time points under salt stress, osmotic (mannitol), cold, and heat stress ( Figure 6). The real-time result indicates that AcCBL1 could be playing a key role in adaption to extreme conditions as Arabidopsis CBL1 contributes to salt, cold, and osmotic stress [29]. Several studies indicate that CBLs contribute to salt stress [6, 29,35]. In Arabidopsis, AtCBL4 with CIPK24, along with Na+/H+ antiporter SOS1, function in exclusion of Na + from the cytoplasm [3,6]. To further validate the hypothesis, we generated transgenic Arabidopsis plants overexpressing AcCBL1 tagged with GFP. Consistently, the overexpression lines also show the increased localization of AcCBL1 in roots under salt stress (Figures 7 and 8b). Previously, AtCBL1 was reported to be induced after wounding [16,54]. Consistent with these reports, we also found that AcCBL1 is associated with wounding. AcCBL1 protein was induced and translocated to the nucleus after wounding (Figure 8a). Moreover, the independent homozygous T3 transgenic lines (designated as Ox1, Ox2, Ox3) showed better performance under salt stress in terms of present root growth (Figure 9a), primary root growth (Figure 9b,c), germination (Figure 9d), fresh weight (Figure 9e), and overall growth phenotype (Figure 9f,g).
Additionally, fungal infection followed by DAB (3,3-diaminobenzidine) staining resulted in the better performance of AcCBL1 overexpressing transgenic plants compared to wild-type plants.
In the presence of peroxidases, DAB is oxidized to generate a dark brown precipitate which is easily monitored to detect the existence and dispersal of hydrogen peroxide in cells [48]. The biotic stress would result in the generation of reactive oxygen species (ROS), resulting in oxidative stress to plants.
After 24 h of fungal stress the transgenic plants displayed less damage to leaves (Figure 10b) and less accumulation of hydrogen peroxide (Figure 10c) than wild-type. In plants, the elevation in the cytosolic calcium (Ca 2+ ) has been previously documented [60]. The AcCBL1 could be sensing the change in the cytosol after salt and wound stress, and relaying the signal downstream would be helping the AcCBL1 overexpressing plant to perform better under stress conditions. Taken together, our results clearly indicate that ectopic expression of AcCBL1 enhances salt and biotic stress tolerance by facilitating scavenging of the Na + and ROS from the cell.

Conclusions
In the present study, a comprehensive analysis of CBL-CIPK gene family was carried out in the economically important pineapple plant. A total of 8 CBL and 21 CIPK proteins were identified and characterized. The expression profile of pineapple CBL-CIPK genes suggest that AcCBL and AcCIPK proteins play key roles in the pineapple growth, development, and response to abiotic stresses. Further, the functional characterization of AcCBL1 shed light on its biological role and contribution during salt stress and wounding. The present study could be a foundation for further research and serve as a basis to utilize CBL-CIPK genes for development of next-generation crops.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/9/7/293/s1, Figure S1: The distribution of conserved motifs in pineapple CBL proteins, Figure S2: The distribution of conserved motifs in pineapple CIPK proteins, Table S3: List of primers used in present study.