Genome-wide identification and characterization of the CKII gene family in the cultivated banana cultivar (Musa spp. cv Tianbaojiao) and the wild banana (Musa itinerans)

Plant casein kinase II (CKII) plays an essential role in regulating plant growth and development, and responses to biotic and abiotic stresses. Here, we report the identification and characterization of the CKII family genes in Musa spp. cv. ‘Tianbaojiao’ (AAA group) and the wild banana (Musa itinerans). The 13 cDNA sequences of the CKII family members were identified both in ‘Tianbaojiao’ and wild banana, respectively. The differences between CKII α and CKII β members are corroborated through the subcellular localizations, phosphorylation sites and gene structures. The cloning of CKII β-like-2 gDNA sequences in wild banana and ‘Tianbaojiao’ and the analysis of gene structures showed MiCKIIβ-like-2b and MaCKIIβ-like-2 are likely alternatively spliced transcripts, which were derived from the alternative splicing events that involved exon deletion. The qPCR validation showed differential expression CKII family members in response to cold stress and also in all tested tissues (leaf, pseudostem and root) of wild banana. In particular, the normal transcript MiCKIIβ-like-2a was highly expressed in response to cold stress in wild banana; oppositely, the alternatively spliced transcript MiCKIIβ-like-2b was quite lowly expressed. The complex origin and long-term evolution of Musa lineage might explain the alternative splicing events of CKII β-like-2.


Introduction
Casein kinase II (CKII or CK2) is a Ser/Thr kinase involved in the regulation of protein functions in eukaryotes. Plant CKII is a tetrameric protein composed of two catalytic (α) and two regulatory (β) subunits, and it is also a pleiotropic enzyme. It plays an essential role in regulating various cellular processes such as growth, development, circadian rhythms, light responses, hormone responses, transcription, translation, cell-cycle regulation, nuclear transport, Ca 2+ storage, seed storage, salicylic acid-mediated defenses, flowering time, DNA repair and responses to biotic and abiotic stresses in plants, such as maize, tobacco, wheat, mustard and a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 genes in banana genomes. They were further analyzed as the candidate CKII genes of banana genomes.

Isolation of the CKII family genes cDNA sequences and CKIIβ-like-2 gDNA sequences from wild banana and 'Tianbaojiao'
The leaves of the wild banana (Musa itinerans) from Sanming City, China and the cultivated banana 'Tianbaojiao' (Musa spp., Cavendish, AAA group, the famous tranditional cultivar in China, which originated from the wild banana Musa acuminate, AA group) collected from the Banana Germplasm Nursery of Institute of Horticultural Biotechnology of Fujian Agriculture and Forestry University were used as the materials for RNA and DNA extraction, according to the method of Feng et al. (2015) [25]. Total RNA was reverse transcribed using a Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Fermentas, EU) for cDNA sequences cloning. Using candidate CKII genes in banana genomes combined with published CKII sequences in NCBI, all of the CKII family members cDNA sequences of wild banana and 'Tianbaojiao' were cloned by RT-PCR (reverse transcription PCR) technique. The CKIIβ-like-2 gDNA sequences of wild banana and 'Tianbaojiao' were obtained from DNA templates using PCR. Primer sequences were designed from known CKII sequences in NCBI and the banana genome databases, and are listed in S1 Table.

Bioinformatic analysis of CKII family genes in the A genome, the wild banana and 'Tianbaojiao'
On the NCBI website, the nucleotide and protein sequences of CKII family members were identified by BLASTn and BLASTp, respectively. DNAMEN 6.0 was used to analyze CDSs (coding sequences) and protein sequences [23][24]. MEGA 6.0 was used to construct the phylogenetic trees of the CKII proteins, and the NJ (neighbor-joining) method was then applied to this analysis with 1,000 bootstrap replications. GSDS was used to analyze the gene structures. The conserved domains were analyzed on the NCBI website. NetPhos 3.1 was used to analyze the CKII phosphorylation sites. The conserved motifs of CKII protein sequences were analyzed on MEME server [26]. The protein subcellular localization prediction tool of 'PSORT' was used to predict the subcellular location of the CKII protein sequences.

Plant materials and treatments for qPCR
The wild banana (Musa itinerans) from Sanming City and the cultivated banana 'Tianbaojiao' (Musa spp., AAA group) were used in this study. The in-vitro plantlets were regenerated by tissue culture from the explants of suckers. After transplanting them to the pots and cultivating for 1 month at 28˚C under 2000 lx throughout lighting in a 12 h/12 h light-dark cycle, seedlings at the uniform growth stage were selected for treatments. After sufficiently watering for 2 d, the seedlings were put in the growth chambers set to 28˚C (the control), and at 13˚C, 4˚C and 0˚C, under 2000 lx fluorescent lighting in a 12 h/12 h light-dark cycle (synchronized with the natural light cycle) at a relative humidity of 70%-80% for 24 h. After 24 h treatments, the first young leaf was detached from 10 seedlings at each temperature point (28˚C, 13˚C, 4˚C and 0˚C) for each biological replicate. The leaf samples of each of the 10 seedlings were harvested and pooled for each temperature point. All of the treatments were performed with 3 biological replicates. Finally, they were frozen in liquid N 2 and stored at -80˚C for total RNA extraction and used in the qPCR (real-time quantitative PCR) assay. The leaves, roots, and pseudo-stems were also to taken from the potted plants grown at 28˚C.

Real-time quantitative PCR and data analysis
The total RNA extracted from the leaves after cold treatments (including the control) using Column Plant RNAOUT 2.0 Kit (TIANDZ, China), and 0.5 ug total RNA was used for reverse transcription of qPCR analysis with PrimeScript™ RT Master Mix (Perfect Real Time) kit (Takara, Japan) according to the method of Feng et al. (2015) [25]. The expression detection of the CKII family genes was performed on a LightCycler 480 (Roche). The reaction system and procedures were those of Feng et al. (2015) [25]. The qPCR analyses were performed as described by Lin and Lai (2013) [27] and the CAC gene was used as the internal control [28]. The primer sequences were designed using Primer 3 input software and are listed in the S2 Table. The amplification efficiency for each primer pairs of the CKII family genes was determined in a qPCR assay using a five-fold dilution series from a pooled cDNA template (S3 Fig). The PCR efficiency values of all CKII family genes ranged from 1.853 to 2.040, and as listed in S2 Table. SPSS was used to assess the statistically significant differences of data, and all data are expressed as the means ± SDs of three independent replicates. Duncan's multiple range test was used for the significant differences. Ã : significant difference (at p-value <0.05) identified by comparing with 28˚C, ÃÃ : very significant difference (at p-value <0.01) identified by comparing with 28˚C.

Analysis of the CKII family genes in banana genomes A and B
In total, there are 13 CKII family genes in banana genome A and 11 CKII family genes in banana genome B. The functional domains analyses indicated that all 13 members of the CKII family in banana genome A contained STKc_CK2_alpha or CK_II_beta functional domains, while the 11 CKII family genes of banana genome B had only three members (ITC1587_Bchr2_P04995, ITC1587_Bchr6_P16283 and ITC1587_Bchr6_P18330) that contained complete functional domains (STKc_CK2_alpha or CK_II_beta). The phylogenetic tree of the 13 CKII members from genome A and the 11 CKII members from genome B had two branches, one containing CKII α members and the other containing CKII β members and unclassified CKII subunits members. Additionally, ITC1587_Bchr9_P25856 and ITC1587_Bchr5_P13181, with PKc-like superfamily domains, were clustered to a clade (S4 Fig). Thus, the integrity and accuracy of CKII from genome A was greater than that from genome B, which was suggested to function as the reference genome for identification of the CKII gene family members in the genus Musa.

Cloning of the CKII gene family members in 'Tianbaojiao' and the wild banana
Using RT-PCR, 13 cDNA sequences of the CKII family members were obtained from the Musa spp. cv. 'Tianbaojiao' and wild banana (Musa itinerans), respectively ( Table 1). The sequence lengths of 9 members were the same but those of the other 4 members were different between the 'Tianbaojiao' and the wild banana (Table 1). Between them, both CKIIβ-4-2 and CKIIβ-4-3 had sequences that differed by 3-6 bp (Fig 1A, Fig 1B and 1C), and CKIIβ-3-like ( Fig 1D) and CKIIα-4 ( Fig 1E) also had sequence differences, making them more similar to those of genome A, which resulted in different stop codons. In addition, there were 2 transcripts of CKIIβ-like-2 in wild banana, and one (MiCKIIβ-like-2a) was similar to that of genome A, while the other (MiCKIIβ-like-2b) was similar to that of the 'Tianbaojiao' banana (MaCKIIβ-like-2) (Fig 1F). The sequence analysis indicated that the MiCKIIβ-like-2a and MiCKIIβ-like-2b genes had a 216 bp sequence difference, which belonged to an exon region when compared with the A genome. Therefore, we inferred that an alternative splicing event during evolution resulted in an exon deletion in the transcripts of MiCKIIβ-like-2b and MaCKIIβ-like-2. The sequence comparisons of CKII family members among wild banana, 'Tianbaojiao' and genome A were conducted ( Table 2). Most of the members were the same or similar among these comparisons, except for the alternatively spliced transcripts of MiCKIIβlike-2b and MaCKIIβ-like-2, which were both quite different from the transcripts of the genome A. In addition, the CKII α subunit members were generally similar, while CKII β subunit members were relatively different.
The analysis of the functional domains indicated that all of the CKII family members contained the whole STKc_CK2_alpha or CK_II_beta conserved domains in wild banana and 'Tianbaojiao'.

Predicted subcellular localizations of the CKII family members among wild banana, 'Tianbaojiao' and the A genome
The subcellular localization of the CKII family members among wild banana, 'Tianbaojiao' and the A genome were predicted ( Table 3).
Comparisons of the subcellular localizations among the CKII family indicated that all of the members, except for MiCKIIβ-like-1 from wild banana, were the same among wild banana, 'Tianbaojiao' and the A genome. Furthermore, all of the CKII β subunit members, except for https://doi.org/10.1371/journal.pone.0200149.t001 CKIIβ-like-1 from 'Tianbaojiao' and the A genome, were localized to the nucleus. However, the subcellular localizations of the CKII α subunit members varied, including cytoplasmic, mitochondrial, and extracellular (including cell wall). In particularly, MaCKIIα-4, Ma06_p36630.1 and MiCKIIα-4 were predicted to localize to the mitochondrial with 100%, 100%, and 95.7% probabilities, respectively, which indicated that CKIIα-4 likely functioned in the mitochondria. The clustering analysis, combined with the subcellular localizations of the CKII family members from the wild banana, 'Tianbaojiao' and the A genome are shown in Fig 2, and both the CKII α and the CKII β members were clearly assigned to two branches. The CKII β members were assigned to one branch (Fig 2A), which were localized to the nucleus except for CKIIβ-like-1 from 'Tianbaojiao' and the A genome. The CKII α members were assigned to another branch, and being further clustered which were consistent with subcellular localizations site, i.e. cytoplasmic (Fig 2B), extracellular, including cell wall ( Fig 2C) and mitochondrial ( Fig 2D).

Analyses CKII family members gene structures among the wild banana, 'Tianbaojiao' and A genome
The gene structures of the CKII family members among wild banana, 'Tianbaojiao' and the A genome were analyzed (Fig 3). The CKII α members contained 10 exons and 9 introns, while all the CKII β members contained 5 exons and 4 introns, except for MiCKIIβ-like-2b from the wild banana and MaCKIIβ-like-2 from 'Tianbaojiao', which contained 4 exons and 3 introns. The differences in these two members may have resulted from an alternative splicing event during evolution that resulted in an exon deletion. The structures define the functions, therefore, the CKII α and β subunit members likely functioned differently.

Predicted occurrence of CKII phosphorylation sites among the wild banana, 'Tianbaojiao' and A genome
CKII phosphorylation sites were predicted among the wild banana, 'Tianbaojiao' and A genome (S3 Table). The results showed that the numbers of phosphorylation sites in the CKII family members among the wild banana, 'Tianbaojiao' and A genome varied from 1 to 12, and the numbers of phosphorylation sites in the CKII β members (except for the proteins from the 2 alternatively spliced transcripts MiCKIIβ-like-2b and MaCKIIβ-like-2) were generally greater than those in the CKII α members. The former were between 5 and 12, and the latter were between 1 and 2, which was in accordance with the differences in the functions of CKII α and CKII β. The CKII α members acted mainly as catalysts of phosphorylation, while the CKII β members were highly conserved and acted mainly in the regulation of enzymatic activities.
The sites and numbers of amino acid residues of CKII phosphorylation in CKIIα-1, CKIIβ-4-4, CKIIα-2, CKIIα-3 and CKIIα-4 members were the same, while the others showed some differences among the wild banana, 'Tianbaojiao' and A genome. For example, the amino acid residue serine 104 in Ma05_p01580.1 and serine 148 in Ma04_p36590.1 were both specific to the A genome, while the amino acid residue serine 10 in MiCKII β-4-3 was specific to wild banana.

Expression levels of the CKII family members in wild banana at different temperatures and in different tissues by qPCR
The expression levels of the CKII family members in wild banana were detected by qPCR in leaf tissue at different temperatures (growth temperature of 28˚C as the control, and the low temperatures of 13˚C for critical growth, 4˚C for chilling and 0˚C for freezing) (Fig 5) and in different tissues (leaves, pseudo-stems and roots) (Fig 6). The expression levels of CKIIβ-like-2a, CKIIα-5 and CKIIβ-4-2 were the highest at 4˚C, especially those of CKIIβ-like-2a and CKIIα-5, which were significantly higher than those of the control, and the expression levels of CKIIβ-like-2a, CKIIα-5 and CKIIβ-4-2 in leaves were also higher than those in pseudo-stems and roots. The expression level of CKIIα-3 was lowest at 4˚C, which was highest in leaf. While the expression level of CKIIα-2 in root was higher than that in leaf (the only member). The expression levels of CKIIα-1, CKIIβ-4-1 and CKIIβ-4-4 were all high, but not significant, at 13˚C. The expression levels of these 3 members in leaves were significantly higher than in the other two tissues, even though they showed similar expression patterns. The expression levels  Genome-wide identification and characterization of CKII gene family in the cultivated and the wild bananas of the 5 members, CKIIβ-like-1, CKIIβ-4-3, CKIIβ-3-like, CKIIα-4 and CKIIβ-like-3, changed remarkably at 2 or 3 of the low temperature points, and the expression patterns of CKIIβ-like-1, CKIIβ-4-3, CKIIα-4 and CKIIβ-like-3 were similar at 28˚C, 13˚C and 4˚C. Interestingly, the expression level of CKIIα-4 at 0˚C was 117 times higher than at 4˚C, and it had a significantly higher expression level in pseudo-stem than that in leaves. It was also the only member that expressed more highly in pseudo-stem than in leaves and roots. CKIIα-2 and CKIIα-4, with root and pseudo-stem specific expression, both belonged to CKII α subunit members and had a sequence similarity of 84.68%, and were expressed relatively higher at 0˚C. This suggested that wild banana could activate not only the CKII members in leaves but also the CKII members in roots and pseudo-stems for cold acclimation in response to the 0˚C cold stress. In addition, alternatively spliced transcript CKIIβ-like-2b was expressed at very low levels or could not be detected at different temperatures and in different tissues (data not shown), which was quite different from the expression patterns of the normal transcript CKIIβ-like-2a. The wild banana of Musa itinerans from Sanming City and A genome from 'DH-Pahang' (Musa accuminata, AA group) of the Malaysian wild banana had the normal transcript CKIIβ-like-2a, but the cultivated 'Tianbaojiao' (AAA group) had only the lowly expressed alternatively spliced transcript CKIIβ-like-2. It might be a key gene related to cold stress. To further validate the expression levels of alternatively spliced transcript CKIIβ-like-2 in response to different temperatures in cultivated banana. For above mentioned reasons that the 'Tianbaojiao' cannot grow well at 4˚C low temperature stress, the expression profile of alternatively spliced transcript CKIIβ-like-2 was performed at 28˚C (control) and 13˚C in 'Tianbaojiao' (S6 Fig). The expression level of CKIIβ-like-2 at 13˚C was lower than those of the control. The growth of 'Tianbaojiao' is retarded or stopped at 13˚C, which is thought to be the critical temperature of growth and relatively low temperature for most banana cultivars in China, while CKIIβ-like-2 was down-regulated expression at 13˚C in 'Tianbaojiao'. So the alternatively spliced transcript CKIIβ-like-2 respond negatively to 13˚C low temperature in 'Tianbaojiao', which is opposite trend for the normal transcript CKIIβ-like-2a at 13˚C low temperature in wild banana.

The function of CKII family members might be different in Musa plants
CKII is considered a tetrameric complex consisting of two catalytic α subunits and two regulatory β subunits [14,[29][30]. The gene structures, phosphorylation sites, and conserved motifs of CKII α and β members were specific in three Musa plants. In addition, most of the CKII β subunit members localized in the nucleus, but the CKII α subunit members varied in Musa plants. However, it is opposite in Arabidopsis. All of the CKII α members, except for the members of Alphacp (localized in chloroplast), localized to the nucleus, but the CKII β members localized to various sites in Arabidopsis [14]. CKII subunits localized frequently to the nucleus and cytosol, but they have also been found in other organelles, such as mitochondria, the endoplasmic reticulum and the external and internal surfaces of the plasma membrane [14,[31][32]. In plants, CKII has been found to localize to the cytosol and the nucleus [16], as well as to the chloroplast in mustard and Arabidopsis [8,14]. In this study, the members of CKII family localized mostly in the nucleus, followed by the cytoplasmic and mitochondrial, and then extracellular sites. The maize CKII α subunit was the first catalytic subunit identified in plants [33], and contains 10 exons separated by 9 introns. Similarly, the CKII α members contained 10 exons and 9 introns in three Musa plants. The characteristic of CKII α subunits are highly conserved among different species were reported [10]. In present study, all the CKII β members contained 5 exons and 4 introns except for the alternatively spliced transcript. All the plant CKII β proteins present the major conserved features described for CKII β subunits from other organisms [34]. This high-degree conservation indicates that the CKII functions may be conserved between the different species. In addition, the conserved motifs analysis of CKII family members in three Musa plants showed the motifs among CKII α or CKII β family members were highly conserved. These above results may further illustrate protein kinase CKII is a ubiquitous and highly conserved Ser/Thr kinase. The numbers of CKII phosphorylation sites in the CKII β members (except for the 2 alternatively spliced transcripts) were generally greater than those in the CKII α members in three Musa plants, and it will be worth investigating relevance of CKII subunit functions.
Expression levels of CKII family members at different temperatures and in different tissues were also different in wild banana. In particularly, the wild banana of Musa itinerans contained both the normal CKIIβ-like-2a transcript and the alternatively spliced CKIIβ-like-2b transcript. Genome A had CKIIβ-like-2a, but the cultivated 'Tianbaojiao' had only alternatively spliced transcript CKIIβ-like-2. The two transcripts of CKIIβ-like-2 had distinct expression levels in response to low temperature. The three Musa plants differ in their responses to environmental stress [20,[35][36][37][38][39], and CKII is involved in various plant developmental processes and in responses to biotic and abiotic stresses [2][3]40]. Plant breeders require access to genetic diversity to satisfy the demands for more and higher quality foods that can be produced in a variable or changing climate, and the crop' wild relatives represent a practical gene pool from which to expand the genetic diversity in crop plants [41]. Ortiza and Swennenb (2014) [42] proposed changing from crossbreeding to the biotechnology-facilitated improvement of banana and plantain. CKIIβ-like-2a may be a target gene for cold resistant in cultivated banana breeding by biotechnology technology.  [23]. After WGD, most (65.4%) of the genes included in the Musa α/β ancestral blocks are singletons and only 10% are retained in four copies, in agreement with the loss of most gene-duplicated copies [44]. Genes are more prone to be co-retained or co-lost after WGD [45]. WGDs have played a major role in angiosperm genome evolution [46]. Alternative splicing, which is common in plants, is a rapidly evolving process after gene duplication [47][48]. Alternative splicing could affect gene regulation, gene function and cause functional divergence between duplicates [47,49]. Moreover, alternative splicing plays a crucial role in defense response of plants [50]. Abiotic stresses are known to cause changes in alternative splicing patterns in plants [51][52]. Plants alter splicing patterns in response to temperature stress [53][54][55][56]. After gene and genome duplication, alternative splicing patterns have diverged considerably in an organ-or stress-specific manner during the evolutionary history of Arabidopsis lineage [49]. These above reports may explain the phenomenon of alternative splicing events occurred in Musa plants. In our study, the alternative splicing events resulted in an exon deletion in both MiCKIIβ-like-2b from the wild banana and MaCKIIβ-like-2 from 'Tianbaojiao'. Both the wild bananas of and genome A had the normal CKIIβ-like-2 transcript, CKIIβ-like-2a, which was highly expressed under cold stress in the wild banana. However, the cultivar 'Tianbaojiao' did not contain CKIIβ-like-2a, and had only the alternatively spliced transcript CKIIβlike-2, which was expressed at very low levels or could not be detected in response to cold stress in wild banana and respond negatively to 13˚C low temperature in 'Tianbaojiao'. Furthermore, from single gene duplication to WGD, gene duplication has occurred throughout eukaryotic evolution and contributed greatly to the duplicated genes [49]. The majority of duplicated genes are retained gain new functions and/or expression patterns (neofunctionalization) [49]. In maize leaf, 13% of homology gene pairs have undergone regulatory neofunctionalization [57]. WGD has formed novel functions genes and altered expression patterns [58]. This may be the factors of the different expression patterns in response to cold stress of the two CKIIβ-like-2 transcripts in wild banana. The character of poorly conserved between duplicated genes by WGD was reported [47]. The two CKIIβ-like-2 transcripts are also not conserved in three Musa plants. Besides, Feng et al. suggested the WGD, segmental duplication and complex transcriptional regulation contributed to the gene expansion and mRNA diversity of the MaSODs by the genome-wide identification of SOD gene family in 'Tianbaojiao' [25]. The alternative splicing events were occurred with high frequency in previous study related Musa plant [59][60][61]. Therefore, the likely factors of alternative splicing events occurred in present study are the complex origin and long-term evolution of Musa lineage.

Conclusions
In this study, based on the banana genome database, the cloning, identification and characterization of the CKII family members in Musa spp. cv. Tianbaojiao (AAA group) and the wild banana (Musa itinerans) were reported. 13 cDNA sequences of the CKII family members were obtained from the 'Tianbaojiao' and the wild banana, respectively. The bioinformatics and qPCR analyses of CKII family members suggested that the function of CKII family members might be different in Musa plants. Furthermore, CKII β-like-2a might be a gene related to cold resistant. In addition, the CKII β-like-2 gDNA sequences in wild banana and 'Tianbaojiao' were obtained, and the analysis of sequences and gene structures showed the MiCKIIβ-like-2b in wild banana and MaCKIIβ-like-2 in 'Tianbaojiao' might be the exon deletion alternative splicing transcripts. The alternative splicing events of CKII β-like-2 may result from the complex origin and evolution of Musa lineage.