Identification of CDPK homologs
A total of 116 candidate CDPK sequences were initially identified from the five species examined, using the CDPK-specific HMMs (Pkinase.hmm and EF-hand_7.hmm) from the Pfam database and searching against the genome data. Among these species, the numbers of CDPK were varied, with 24, 28, 16, 14, and 34 members from apple (M. domestica), pear (P. bretschneideri), peach (P. persica), strawberry (F. vesca), and Arabidopsis (A. thaliana), respectively. As listed in Table 1, the CDPKs are composed of amino acids ranged from 323 (AT1G76040) ~ 847 (HF10630), with molecular mass from 37.16 ~ 96.22 kDa and pI from 4.49 ~ 9.90.
The identified CDPK genes are unevenly distributed on individual genomes (Table 1 and Fig. 1). The 24 apple CDPKs (MdCDPKs) are distributed among 12 out of 18 chromosomes, including chromosomes no.2 ~ no.7, no.9 ~ no.12, no.14, and no.17, (Fig. 1A). Each of five apple chromosomes (no.3, no.5, no.10, no.12, no.14) contains three CDPKs, with the remaining chromosomes having one or two members, respectively. The 28 pear CDPKs (PbCDPKs) are distributed among 12 out of 17 chromosomes, including chromosomes no.2 ~ no.6, no.9 ~ no.14, and no.17, (Fig. 1B). Among these chromosomes, chromosomes no.12 and no.10 have the most CDPKs (five and four, respectively), whereas others have various members ranged from one to three. The 14 strawberry CDPKs (FvCDPKs) are distributed among 6 out of 7 chromosomes (Fvb2 ~ Fvb7), without CDPKs on its chromosome no.1 (Fig. 1C). Chromosome no.6 has the most CDPKs (5), in contrast to chromosomes no.4, no.5, and no.7 with the least (1). The 16 peach CDPKs (PpCDPKs) are distributed throughout all its eight chromosomes (Pp01 ~ 08, Fig. 1D), with the CDPK members ranged from one (chromosomes no.2, no.3 and no.6) to four (chromosome no.4). Similarly, the 34 Arabidopsis CDPKs (AtCDPKs) are found throughout all its five chromosomes (Fig. 1E), with the most CDPKs (11) on chromosome no.4 and the least (4) on chromosome no.3. There is a significant uneven distribution of CDPKs on the chromosome no.5, due to clustering of six CDPKs on its short arm (Fig. 1E).
Phylogenetic and gene structural analysis of the CDPKs
To investigate the phylogenetic relationships and molecular evolutionary history of the sequences in the examined species, following the alignment of 116 CDPK proteins, a phylogenetic analysis was conducted and a phylogenetic tree was generated using the Maximum Likelihood (ML) method. The phylogenetic tree showed that the 116 CDPKs were clustered into five main subgroups, among which the highest numbers of members were 33 in subgroups I and IV, followed by 31 and 16 in subgroups III and II, respectively (Fig. 2). And subgroup V has the least members (3), all of which are from AtCDPKs. As shown in Fig. 2, the CDPKs from individual species were grouped into different clades rather than a single one. Additionally, their numbers varied within different subgroups. Out of 24 MdCDPKs, nine were located in subgroup I, seven in subgroups IV, and four in subgroups II and III, respectively (Fig. 2). The other Rosaceae species also exhibited similar patterns in their CDPK distributions, among which 9, 4, and 4 members from pear, strawberry, and peach, were included into subgroup I, respectively. Accordingly, 5, 2, and 2 in subgroup II; 5, 3, and 6 in subgroup III; 9, 5, and 4 in subgroup (Fig. 2). 34 AtCDPKs were dispersed across subgroups I~V, with the most (13) in subgroup III and the least (3) in subgroups II and V (Fig. 2). Additionally, it appears that AtCDPKs were clustered with each other in the five subgroups, compared to the CDPK clustering across Rosaceae species. Moreover, three AtCDPKs were grouped into distinct the subgroup V separated from CDPK homologs from the other species examined (Fig. 2).
Based on sequence alignment, it was found out that all of characteristic domains of CDPK family (i.e. a domain of protein kinase for CDPK activities and four EF-hands for calcium-binding) were presented among 113 out of 116 CDPKs (Fig. 2). Among the remaining 3 CDPKs, AT2G35890 and HF28950 have only two EF-hands (i.e. the 1st and the 2nd ones), whereas Pb001308, without the 1st one, has three EF-hands at the C-terminus, respectively (Fig. 2 and Fig. 3). And there showed a high conservation among the EF-hand domains of the CDPKs identified (Fig. 3).
To characterize their gene structural diversity, the exon-intron organizations of the CDPKs were analyzed (Fig. 4A). The number of exons was diverse, with a minimum of one (i.e. HF00526, HF28950, Pbr027545, Pbr033411, Pbr033416, and Prupe.3G035400) and a maximum of 12 (AT2G17890, AT4G04710, AT4G36070, and AT5G66210). Generally, the CDPK gene structures within each subgroup of the phylogenetic tree, showed a similar pattern, supporting their phylogenetic relationships (Fig. 4A). An exception is that the six CDPKs with a single exon are clustered into a distinct clade within subgroup II, which consists of other CDPKs with seven exons (8 members), six exons (1member), or two exons (1 member). However, CDPKs from a specific clade within a subgroup, apparently have the same numbers of exons (Fig. 4A). In addition, motif analysis by MEME demonstrated that most representatives of the motifs in CDPKs from the same subgroup, showed a conservation in both motif distribution and composition, coordinating with their distribution across various subgroups in the phylogenetic tree (Fig. 4B).
Collinearity analysis of CDPKs
To investigate the gene duplication that promotes the evolution of CDPK gene family among the species examined, multiple-round analysis of collinearity relationship was carried out between each pair of species. A total of 245 CDPK gene-pairs with collinearity relationships were identified, consisting of 36 intraspecies-pairs and 209 interspecies-pairs across each pair of species (Table 2, Fig. 5, Additional file 1: Table S1 and Additional file 2: Figure S1). Among 36 gene-pairs with intraspecies collinearity, 10, 13, 1, and 12 ones were blasted out from apple, pear, peach, and Arabidopsis, respectively (Fig. 5A, 5B and 5C, Table 2 and Additional file 1: Table S1). And no intraspecies-pair of CDPKs was found in strawberry (Fig. 5D). Among 209 gene-pairs with interspecies collinearity, there existed 22, 36, 21, and 25 ones between apple and four other species (i.e. Arabidopsis, pear, strawberry, and peach), respectively (Fig. 5, Table 2 and Additional file 1: Table S1). Both pictorial micro-synteny of 10 MdCDPK gene-pairs and 22 CDPK gene-pairs between apple and Arabidopsis (Fig. 6), were demonstrated to support their synteny relationships.
Apart from apple and Arabidopsis, CDPKs collinearity were also identified between the other species (Additional file 1: Table S1 and Additional file 2: Figure S1). Noticeably, the individual CDPK gene-pairs with collinearity relationships were not only distributed within the same phylogenetic subgroups, but also identical in their exon-intron patterns (Fig. 2 and Fig. 4), such as HF05471-Pbr001322, HF20170-Pbr010307, and AT3G10660-AT5G04870 in subgroup I; Pbr033416-Pbr033411, AT1G35670-AT4G09570, and HF39191-Pbr040137 in subgroup II; Pbr024654-Prupe.5G110500, HF17744-Pbr031892, and HF04323-Pbr039714 in subgroup III; HF15429-Pbr021635, HF01706-Pbr036114, and gene25220-Prupe.7G064300 in subgroup IV; AT2G17890-AT4G36070, AT2G17890-AT5G66210, and AT4G36070-AT5G66210 in subgroup V. This result supported the evolutionary relationships between the identified CDPKs. To assess the evolutionary rates among these CDPK gene-pairs, the Ka (nonsynonymous nucleotide substitutions) to Ks (synonymous nucleotide substitutions) ratios were calculated (Table 2 and Additional file 1: Table S1). The Ka/Ks values ranged from 0.022 to 0.751 for the gene-pairs between apple and other species, while from 0.085 to 0.399 for those within apple. It is noticeable that there is no gene-pair with Ka/Ks values >= 1, inferring that the duplicated CDPKs within the species examined have been undergone purifying selection.
Quantitative analysis of MdCDPKs expression during apple fruit development
To examined the expression patterns of apple MdCDPKs, the expression data set, based on the transcriptomic analysis of two apple strains at their four stages of fruit development , was applied to addressing this question. After the quantitative analysis of transcriptomic data, the whole expression levels of 24 MdCDPKs were visualized via heatmap plotting (Fig. 7). As showed in Fig. 7, five out of 24 MdCDPKs (i.e. HF03960, HF05458, HF13700, HF20185, and HF29516) presented no any expression amounts at the transcriptional level. In contrast, the remaining 19 MdCDPKs were constitutively expressed with various patterns in related to both the apple strains (BLO-yellow fruit skin vs. KID-red fruit skin) and the different stages of fruit development (S1~S4, Fig. 7). The expansion patterns of these MdCDPKs were in the trends with three types: (I) higher expression levels, (II) lower expression levels throughout four stages of fruit development, and (III) apparent difference in expression levels at the different stages of fruit development. The expression of six MdCDPKs (HF00526, HF05266, HF10624, HF17744, HF36202, and HF39191) were characterized with higher expression levels (i.e. pattern I), while three MdCDPKs (HF04060, HF20170, and HF28950) were expressed at lower levels (pattern II). Ten other MdCDPKs (HF01706, HF04323, HF05471, HF06540, HF09216, HF10630, HF13801, HF14253, HF15429, and HF39491) showed the difference in expression levels at four stages of fruit development (i.e. pattern III), among which the expression levels of HF01706, HF04323, HF09216, and HF39491, were in a trend of gradual elevation from the stage S1 to S4, with expression peaks at the stage S4 for both of the apple strains (i.e. BLO and KID). On the contrary, the transcriptional expression in a reverse pattern were found out from another group of MdCDPKs (HF05471, HF06540, HF10630, HF13801, HF14253, and HF15429), with higher amounts at the S1, compared to those at other stages (Fig. 7).
To further validate if these MdCDPKs were differentially expressed with significance among the four stages of fruit development, or the two apple strains, the transcriptional data were screened for the MdCDPKs by the criterion: log2FoldChange > 1 and p-value < 0.05 in the present study. As a result, the MdCDPKs, differentially expressed with significance, were presented under the twelve types of comparison (Fig. 8). For both the apple strains BLO (Fig. 8A, 8C and 8E) and KID (Fig. 8J, 8K and 8L), the up-regulatory genes (HF05266 and HF09216) and down-regulatory genes (HF05471 and HF15429) were differentially expressed with significance under the comparison S4 vs. S1, S4 vs. S2, and S4 vs. S3. Interestingly, irrespective of their difference in expression fold-changes, the significantly up-regulatory genes (HF05266 and HF09216) and down-regulatory genes (HF05471 and HF15429), were also found out by inter-strain comparison, including KID_S4 vs. BLO_S1, BLO_S2, and BLO_S3 (Fig. 8B, 8D and 8F), or BLO_S4 vs. KID_S1, KID_S2, and KID_S3 (Fig. 8G, 8H and 8I), respectively. In addition, the significantly down-regulatory gene HF20170 was presented under the comparison S4 vs. S2 for KID (Fig. 8K) or KID_S4 vs. BLO_S1 (Fig. 8B), KID_S4 vs. BLO_S2 (Fig. 8D), while the significantly up-regulatory HF00526, HF04323, and HF01706 were under the comparison S4 vs. S2 for BLO (Fig. 8C), S4 vs. S2 for KID (Fig. 8K), and S4 vs. S3 for KID (Fig. 8L), respectively.
Therefore, among the MdCDPKs with the expression pattern III at transcriptional level (Fig. 7), five members (HF01706, HF04323, HF05471, HF09216, HF15429) showed significantly differential expression between the specific stages of fruit development. And the previous described MdCDPKs with the expression pattern I (HF00526, HF05266) or II (HF20170) should be sorted to the pattern III, though these CDPKs appeared the higher or lower expression amounts throughout the four stages of fruit development due to the limited resolution by the heatmap (Fig. 7).