Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum

As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.


Introduction
In their natural habitat, plants frequently encounter different abiotic and biotic stresses, and have evolved a complicated defense system to defend themselves against such stresses. It is crucial for plants to recognize and perceive stress in order to comprehensively reprogram their metabolism and switch to defense mode. Upon challenge with biotic or abiotic stresses, Ca 2+ flux across the plasma membrane is generally triggered, leading to a rapid increase in the cytoplasmic Ca 2+ concentration with varying frequency, amplitude, and duration dependent on the stress cues. These calcium concentration changes are sensed and decoded by different Ca 2+ sensors/Ca 2+ -binding proteins, including calmodulins (CaMs), calmodulin-like proteins (CaMLs), calcineurin Blike proteins (CBLs), and calcium-dependent protein kinases (CDPKs), which subsequently result in different downstream defense responses. However, the underlying mechanism remains largely unknown.
CDPKs are encoded by a large gene family: genome-wide analyses have identified 34 CDPK genes in Arabidopsis (Cheng et al., 2002), 31 in rice (Asano et al., 2005;Ray et al., 2007), 40 in maize , 20 in wheat (Li et al., 2008), 30 in Populus (Zuo et al., 2013), and 41 in cotton . Members of the CDPK family differ in their sequences, expression patterns, and subcellular targeting, and can be subdivided into four distinct subfamilies (Frattini et al., 1999;Dammann et al., 2003;Ray et al., 2007;Wan et al., 2007;Zuo et al., 2013); members within the same subfamily shared similar intronexon organization (Zuo et al., 2013;Liu et al., 2014). However, diversity in the CDPK family among different plant species was also observed. For example, less than half of the Arabidopsis CDPK genes had Populus homologs. In contrast, generally two or more Populus homologs were found for each Arabidopsis CDPK gene (Zuo et al., 2013).
Pepper (Capsicum annuum) is a vegetable crop of great agricultural and economical importance, and is also a typical species of Solanaceae with soil-borne diseases. Heavy losses in pepper production are frequently caused by various soil-borne diseases as well as different abiotic stresses such as drought, extreme temperatures, and salinity. Understanding the molecular mechanisms underlying resistance/tolerance to diseases or stress is beneficial in engineering improved crop disease resistance or stress tolerance, and functional characterization of CDPKs is a feasible approach to dissect the these molecular mechanisms. Recent genetic evidence indicates that plant CDPK genes transcriptionally upregulated during biotic and abiotic stress responses can have important roles in these stresses resistance or tolerance (Chico et al., 2002;Asano et al., 2012;Fu et al., 2013;Jiang et al., 2013;Wei et al., 2014), for example, OsCPK9 transcription was induced by abscisic acid (ABA), PEG6000, and NaCl treatments, and OsCPK9 overexpression enhanced., while OsCPK9 RNA interference decreased drought stress tolerance (Wei et al., 2014), and the expression of OsCPK10 was strongly induced following treatment with a Magnaporthe grisea elicitor and rice OsCPK10 acts a crucial regulator in plant immune responses (Fu et al., 2013). To get a comprehensive understanding of CDKPs in pepper and their possible involvements in pepper's stress responses, a genomewide identification and expression analysis of CDPKs was performed in the present study, and a total of 31 CDPK genes and 5 CRK genes were found. These CDPKs were grouped based on their phylogenetic relationships into four subgroups and were located to specific chromosomes. The transcriptional expression of CDPK genes in pepper plants against salt, heat treatment, and Ralstonia solanacearum inoculation was also analyzed.

Materials and Methods
Database Search for CDPK Genes in Pepper, Phylogenetic Analysis, and Structural Divergence The protein sequences for 34 Arabidopsis CDPKs were obtained from the Arabidopsis Information Resource (http://www. Arabidopsis.org/). Sequences from the pepper genome database were downloaded from http://peppersequence.genomics.cn/ page/species/index.jsp. For the identification of the pepper CDPK gene family, Arabidopsis CDPKs protein sequences were used to search the pepper genome and NCBI databases using BLASTP. Multiple alignments of amino acid sequences were aligned using Clustal X (Zhou et al., 2009). A phylogenetic tree was created according to the neighbor-joining method using the MEGA5.0 program (Tamura et al., 2011). The gene structures were displayed using an online tool (http://gsds.cbi.pku.edu.cn/). cDNA sequences and gene sequences were downloaded from this website: http://peppersequence.genomics.cn.
The seeds of pepper GZ03 were sown in a soil mix [peat moss:perlite, 2:1 (v/v)] in plastic pots and were placed in a greenhouse at 25 • C, 60-70 mmol photons m −2 s −1 , a relative humidity of 70%, and a 16-h light/8-h dark photoperiod.

Pathogens and Inoculation Procedures
R. solanacearum strain FJC100301 was isolated previously in our lab and amplified according to the method of Dang et al. (2013). The bacterial cell solution was diluted to 10 8 cfu mL −1 (OD 600 = 0.8) with 10 mM MgCl 2 . Pepper plants were inoculated by infiltrating 100 µL of the resulting R. solanacearum suspension into the third leaves from the apical meristem using a syringe with a needle. The leaves were harvested at the indicated time points for the preparation of total RNA.

Salt Stress and High Temperature Treatments
For salt stress treatment, eight-leaves pepper plants were treated with 250 mM NaCl and the plants with water as control. The salttreated plants and the control plants were placed in a greenhouse under the same conditions mentioned above. The leaves were harvested at the indicated time points for the preparation of RNA. High temperature treatment was carried out in a growth chamber a,b the gene accession numbers from Chiltepin and CM334. The nomenclature of Chiltepin for genes is Capang**g######, respectively, in which "Capang" is the short name of Capsicum annuum var. glabriusculum. "**" represents the number of chromosomes; "g" represents genome; "######" is the identifier for a gene that is anchored to a certain chromosome.
Frontiers in Plant Science | www.frontiersin.org at 43 • C, 100% humidity, 60-70 mmol photons m −2 s −1 , and a 16-h light/8-h dark photoperiod. Normal pepper plants that were not exposed to salt stress and high temperature treatment were used as controls. The leaves were harvested at the indicated time points for the preparation of total RNA.

Quantitative Real-time PCR
To determine the relative transcript levels of selected genes, realtime PCR was performed with specific primers (Table S1 for genespecific primers) according to the manufacturer's instructions for the Bio-Rad Real-time PCR system (Foster City, CA, USA) and the SYBR Premix Ex Taq II system (TaKaRa Perfect Real Time). Total RNA was isolated from pepper plants using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reverse-transcribed using the PrimeScript RT-PCR kit (TaKaRa, Dalian, China). A 10-fold dilution of the resulting cDNA was amplified using the SYBR Premix Ex Taq II kit and the Bio-Rad Real-time PCR system in a 10-µL volume with the following program: 95 • C for 30 s; 40 cycles of 95 • C for 5 s and 60 • C for 34 s; 95 • C for 15 s. Amplification of the target genes was monitored during each cycle by SYBR green fluorescence. The Ct (threshold cycle), defined as the real-time PCR cycle at which a statistically significant increase in reporter fluorescence was first detected, was used as a measure for the starting copy numbers of the target genes. Three replicates of each experiment were performed. Data were analyzed by the Livak method (Livak and Schmittgen, 2001) and expressed as a normalized relative expression level (2 − CT ) of the respective genes. The relative transcript levels of the analyzed pepper genes were normalized to the transcript levels of CaActin. In each case, three technical replicates were performed for each of at least three independent biological replicates.

Identification of CDPK and CDPK-Related Protein Kinase (CRK) Genes in Pepper Genome
The recently published genome of C. annuum varieties CM334 and Chiltepin make it possible to identify all pepper CDPK genes (Kim et al., 2014a). Blast searches of the pepper genome of CM334 were performed by using all of the 34 CDPK sequences in Arabidopsis as baits, and a total of 31 putative CDPKs and 5 FIGURE 1 | Differences in the deduced amino acid sequences of five CDPK genes between pepper cultivars Chiltepin and CM334. Chiltepin-CDPK: the deduced amino acid sequences of CDPK genes from Chiltepin. CM334-CDPK: the deduced amino acid sequences of CDPK genes from CM334. The red box: the detailed differences in the amino acid.
Frontiers in Plant Science | www.frontiersin.org CDPK-related genes were identified (  (Harper et al., 1991;Cheng et al., 2002;Harmon, 2003). In addition, all the pepper CDPK genes were predicted to have four EF-hand motifs in the CaM-like domain, which can recognize and bind Ca 2+ molecules ( Table 1, Cheng et al., 2002;Hrabak et al., 2003). The N-terminus of a subset of CDPK proteins contain a myristoylation motif, which is believed to promote protein-membrane and protein-protein interactions (Martín and Busconi, 2000;Hrabak et al., 2003). It has been reported that proteins containing myristoylation motifs tend to localize in the plasma membrane Martín and Busconi, 2000). Among the identified 31 pepper CDPK proteins, 11 CDPKs were predicted to contain myristoylation motifs at their N-termini. Additionally, five CDPK-related protein kinase genes (CRKs) were identified in the pepper genome, less than the eight CRKs FIGURE 2 | Phylogenetic tree of CDPK and CRK genes from genomes of pepper and Arabidopsis. A neighbor-joining tree was constructed using the MEGA5.0 program with 1000 bootstrap analyses using the full length amino acid sequences of 31 pepper CDPK genes, five pepper CRK genes, 34 Arabidopsis CDPK genes, and eight Arabidopsis CRK genes. The CDPK genes were classified into four subfamilies (groups I, II, III, and IV). The CRK genes were classified into two subfamilies (CRK I and CRK II).
Frontiers in Plant Science | www.frontiersin.org identified in Arabidopsis . These pepper CRKs range in molecular mass from 55.9 to 67.3 kDa, and their structure is similar to that of CDPKs except for the degenerate calmodulin-like domains. Consistent with previous findings that most CRKs are modified at the N-terminus Asano et al., 2005), all the pepper CRKs except CaCRK5 contained potential myristoylation sites at their Ntermini (Table 1). CDPKs in the pepper variety Chiltepin were also identified and CDPKs in the two pepper germplasms were compared. The sequences of the majority of the CDPK family members in the two germplasms were the same, except in the case of CaCDPK6, CaCDPK7, CaCDPK10, CaCDPK12, and CaCDPK14, in which different sequences of the same gene were found in the two germplasms (Figure 1). Moreover, CaCDPK31, which is present in CM334, was not found in Chiltepin.

Phylogenetic Analysis of CDPK and CRK Genes and Their Chromosomal Location
To examine the phylogenetic relationship among the CDPKs and CRKs in pepper, the CDPKs of Arabidopsis and pepper, were constructed from alignments of the full-length kinase sequences using MEGA5.0. Similar to the previous studies in Arabidopsis, CDPK genes in pepper were grouped into four subfamilies, with 12, eight, seven, and two members in subfamily I, II, III, and IV, respectively (Figure 2 and Table 2). Similar to the CDPKs in Arabidopsis, Populus, rice, and cotton, subfamilies I and II in pepper consist of large numbers of CDPKs, which exhibit a great diversity in number among different plant species: for example, four more CDPKs in subfamily I were found in cotton than in Arabidopsis, and seven more CDPKs in subfamily II were found in cotton than in poplar and rice. The members in subfamily III and IV were approximately identical across the five plant species, and subfamily IV contained the lowest number of CDPKs. Five CRKs were found in rice and pepper, whereas in Arabidopsis and Populus trichocarpa, eight and nine CRKs were found, respectively. These results indicated that differences in the number of CDPK genes in different plant species might be contributed largely by the numbers of CDPK I or CDPK II that have evolved independently among the different organisms.
To obtain further insights into the possible structural evolution of CDPK genes in the pepper genome, diverse exonintron organizations of CaCDPKs were compared. As shown in Figure 3 and Table 3, CaCDPKs clustered in the same subfamily shared very similar exon-intron structures. Most members of subfamily I possessed seven exons, with the exception of CaCDPK2 and CaCDPK17 (12 exons). Members in subfamily II harbored diverse numbers of exons: eight exons were found in CaCDPK3, CaCDPK5, CaCDPK10, and CaCDPK11, seven exons were found in CaCDPK7, CaCDPK23, CaCDPK26, and CaCDPK27, and five exons were found in CaCDPK14. In subfamily III, CaCDPK16, and CaCDPK18 had nine exons, CaCDPK1, CaCDPK24, and CaCDPK25 had eight exons, and CaCDPK9 had seven exons. The two members in subfamily IV both had 12 exons. The results indicate that CDPK genes with higher homogenous sequences tend to have the same numbers of exons, interestingly, members of subfamily I and subfamily IV have the same numbers of exons in pepper and cotton.  Among the 31 pepper CDPKs, 28 genes were mapped onto the chromosomes ( Table 1). The largest number of CDPK genes (six) were located to chromosome 1, four CDPK genes were located to chromosome 4, and chromosomes 7 and 12 were found to harbor three CDPK genes each. Chromosome 2, 3, 8, and 12 were each found to harbor two CDPK genes. One CDPK gene was found on chromosomes 5, 10, and 11. No CDPK gene was mapped to Chromosome 9. Three genes (CaCDPK29, CaCDPK30, and CaCDPK31) could not be placed on any chromosome; these may belong to some gap regions in pseudomolecules. The five CRK genes were also mapped to different chromosomes, Chromosome 2 and 3 were found to harbor two CRK genes each, whereas one CRK gene was found on chromosome 1 ( Table 1).

Pepper CDPK Genes and CRK Genes are expressed in Different Tissues in Pepper Plants
Extensive studies have suggested a close relationship between gene expression and function. To investigate the possible roles of the CDPK genes in the pepper genome, the expression profiles of the 36 CDPK and CRK genes were analyzed in 17 tissues and developmental stages using microarray expression data recently published by Kim et al from CM334 pepper plants (Figure 4, Kim et al., 2014b). The results showed that most of the CDPK and CRK genes were expressed in different tissues and developmental stages in pepper. In addition, we analyzed the expression of all the pepper CDPK and CRK genes in different organs such as root, stem, leaf, flower, and fruit in plants of GZ03, an inbred line of pepper, under normal conditions using qRT-PCR (Figure 5). All of the CDPK genes except CaCDPK16, and all of CRK genes in the pepper genome, were expressed in at least one of the five tissues in non-stressed pepper plants, whereas CaCDPK16 was not expressed in any of the five organs in non-stressed pepper plants. CaCDPK22 was expressed constitutively in roots, but its expression levels in other organs were very low. CaCDPK2, CaCDPK3, CaCDPK4, and CaCDPK31 were expressed constitutively only in flowers. All these data indicate that the members of the CDPK gene family and CRK genes might be involved in the growth and development of different tissues or organs of pepper plants.

Expression Profiles of Pepper CDPK and CRK Genes under High Salt Stress
Salt stress is a major abiotic stress that exerts detrimental effects on crop growth and development, and causes heavy losses in crop yield. To test if CDPK genes in the pepper genome are involved in the response of pepper to salt stress, we examined the expression patterns of all the CDPKs and CRKs in pepper under salt stress treatment by qRT-PCR (Figure 6). Among the 31 CDPK and five CRK genes, 10 CDPK genes showed transcriptionally modified expression in response to salt stress, and all of the five CRK genes did not respond to salt stress. These 10 salt-responsive CDPK genes belonged to groups I (five genes), II (four genes), and III (one gene). It is noticeable that none of the salt stress-inducible CDPKs belong to group IV, suggesting that the response of pepper to salt stress might involve the reprogramming of multiple processes mediated by different CDPK genes. Similar expression profiles were found between CaCDPK6, CaCDPK12, and CaCDPK23, between CaCDPK20 and CaCDPK21, between CaCDPK11 and CaCDPK19, and between CaCDPK7 and CaCDPK25 under salt treatment, implying that multiple CDPK genes may act in a

Expression Profiles of Pepper CDPK Genes and CRK Genes under High Temperature Stress
High temperature is another important abiotic stress that also exerts detrimental effects on crop growth and development and causes heavy losses in crop yield. To investigate if CDPK and CRK genes are involved in the response of pepper to heat stress, we examined their expression patterns by qRT-PCR under heat stress treatment. Out of the 35 CDPK and CRK genes, 13 CDPK and two CRK genes showed modified expression profiles in response to high temperature, suggesting that the response of pepper to high temperature may also include the reprogramming of multiple processes mediated by different CDPK and CRK genes. As showed in Figure 7, similar expression profiles among CaCDPK25, CaCDPK13, and CaCDPK5, CaCDPK30, CaCDPK23 and CaCRK3, CaCDPK18, CaCDPK20, and CaCDPK21, and between CaCDPK1 and CaCDPK4, CaCRK4, and CaCRK5, CaCDPK31, and CaCDPK17 were found in response to high temperature treatment, implying that more than one CDPK genes may act in a coordinated fashion to mediate a single process in pepper's response to high temperature stress. The results also showed that most of the high temperature-responsive CDPK genes were upregulated in response to high temperature treatment; however, the transcripts of CaCDPK18, CaCDPK20, and CaCDPK21 decreased at 2-6 hpt (hours post-treatment) and increased at 12-24 hpt, CaCRK4, and CaCRK5 were downregulated in response to high temperature treatment, suggesting that these CDPK genes are involved in the response of pepper to high temperature, acting as positive or negative regulators.

Expression Profiles of Pepper CDPK Genes and CRK Genes under R. solanacearum Inoculation
To test whether pepper CDPK and CRK genes are involved in the response to biotic stresses, the expression profiles of CDPK and CRK genes were monitored in pepper plants challenged with R. solanacearum, an important causal agent of pepper bacterial wilt worldwide. The results showed that, out of the 35 CDPK and five CRK genes in the pepper genome, 8 CDPK genes and one CRK gene showed altered expression patterns in response to R. solanacearum inoculation. As shown in Figure 8, eight CDPK genes were upregulated upon R. solanacearum inoculation, and only CaCDPK10 was downregulated upon R. solanacearum inoculation. Among the eight upregulated CDPK genes, CaCDPK27, and CaCDPK22 were dramatically induced by R. solanacearum inoculation. Similar expression profiles were found among CaCDPK8, CaCDPK26, and CaCDPK31, CaCDPK15, CaCDPK17, and CaCRK4, respectively, implying that these genes may act in a coordinated manner in mediating the response of pepper to R. solanacearum inoculation.

Discussion
As typical member of Solanaceae and an agriculturally important vegetable, pepper is characterized by its large genome (∼2.7 Gb), which is much larger than that of Arabidopsis (125 Mb) and rice (389 Mb). The 31 CDPK genes are much less than the number expected, compared to 34 CDPK genes in Arabidopsis (Cheng et al., 2002) and 31 in rice (Asano et al., 2005;Ray et al., 2007). Similar to the CDPK genes in other plant species such as Arabidopsis, rice, poplar, and cotton, the 31 CDPK genes were grouped into four subfamilies, with similar numbers of CDPK III and IV, and diverse numbers of CDPK I and II genes compared to those in other plant species, supporting the notion that the different numbers of CDPK genes in different plant species is contributed largely by the numbers of CDPK I or CDPK II genes that have evolved independently in different organisms . Like CDPK genes in other plants such as cotton and maize (Ferreira Neto et al., 2013;Zuo et al., 2013;Liu et al., 2014), the 31 CDPK genes and the five CRK genes identified in pepper genome are unevenly distribute on different chromosomes. Except for the typical CDPK domains, diversities in members of CDPK family among different plant species were observed. For example, less than half of the Arabidopsis CDPK genes had Populus homologs (Zuo et al., 2013). Similar sequences and exon-intron organization patterns were seen to be shared by members of the same subfamily of CDPK genes in the pepper genome and other plant species, suggesting the same origins of CDPK genes in different plant species. Similar to the duplicated CDPK gene pairs found in cotton and other plant species , eight duplicated CDPK gene pairs were found in the genome of pepper. Because more than 81% (∼2.7 Gb) of the pepper genome was composed of different transposable elements (TEs), and the pepper genome is believed to have been expanded by a rapid amplification of retrotransposon elements (Kim et al., 2014b;Qin et al., 2014), the members of the CDPK family might have been expanded by amplification mediated by retrotransposon elements. Additionally, significant differences were observed in the deduced amino acid sequences of CaCDPK6, CaCDPK7, CaCDPK10, CaCDPK12, and CaCDPK14 between CM334 and Chiltepin, and CaCDPK31, which is present in CM334, was not found in Chiltepin, suggesting active diversification of the members of CDPK family among different pepper germplasms. Further functional identification of these diversified CDPK genes might provide new insights into pepper immunity and other biological processes. Accumulation evidences indicate that plant growth, development, and response to environmental stress are largely regulated at transcriptional level (Baena-González et al., 2007;Baena-González and Sheen, 2008;Rymen and Sugimoto, 2012;Buscaill and Rivas, 2014). In the present study, our data showed that CDPK genes in the pepper genome also exhibit specific spatial transcriptional expression patterns in different organs of pepper plants, and some CDPK genes in the pepper genome were found to be transcriptionally modified by cold, high temperature, and R. solanacearum inoculation, similarly to that in other plant species such as maize , cotton , and rice (Ray et al., 2007;Ye et al., 2009), suggesting the involvement of these CDPK genes in plant growth and responses to cold, heat stress, and pathogens. Interestingly, similar responses to salt stress were found in pepper CDPK genes and their orthologues in Arabidopsis. For example, salt stress-responsive CaCDPK31 shared 60% sequence identity to AtCPK11, a positive regulator of salt stress tolerance (Zhu et al., 2007). CaCDPK19 and CaCDPK12 shared 56% sequence identities with AtCPK4, which has previously been shown to be involved in salt stress (Zhu et al., 2007). CaCDPK23 shared 70.89% sequence identity to AtCPK6, whose overexpression enhanced the tolerance of transgenic plants to salt/drought stresses, whereas atcpk6 mutant plants display no obvious phenotypes (Xu et al., 2010). CaCDPK7 shared 58.22% sequence identity to AtCPK21/23, and atcpk21, and atcpk23 mutants showed increased tolerance to hyperosmotic stress, drought, and salt stresses (Ma and Wu, 2007;Franz et al., 2011). These results suggest a close relationship between the sequence of CDPK genes and their function in the salt stress response. Transcriptional reprogrammings were previously assayed comparatively against different biotic and abiotic stresses using stress resistant or tolerant and stress sensitive plant accessions, differences observed between the resistant or tolerant and sensitive plant accessions against the same stress are usually temporal and quantitative not qualitative (Maleck et al., 2000;Tao et al., 2003;Puckette et al., 2008;Tsuda et al., 2009;Thomma et al., 2011;Pons Puig et al., 2015), especially between compatible and incompatible plant-pathogen interactions or ETI (effector triggered immunity) and PTI (PAMP triggered immunity) (Maleck et al., 2000;Tao et al., 2003;Thomma et al., 2011), although the defense reactions are usually faster and stronger in ETI or incompatible plant-pathogen interactions than in PTI or compatible plant-pathogen interactions. The overexpression of CDPK genes such as OsCPK9 (Wei et al., 2014), OsCPK12 (Asano et al., 2012), and ZmCPK4  transcripionally upregulated generally leads to enhanced stress tolerance/resistance in transgenic plants, suggesting potential values of the CDPK genes upregulated by stresses in the present study in genetic improvement of crop stress resistance/tolerance. There were several novel findings in the present study that are worth pointing out. First, upon exposure to cold, heat stress and R. solanacearum inoculation, the expression of 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and R. solanacearum inoculation, respectively. Similar observations were previously published in rice, in which more than one CDPKs such as OsCPK4, OsCPK12, and OsCPK21 were seen to function as positive regulators of the salt response (Asano et al., 2011(Asano et al., , 2012Campo et al., 2014), and OsCDPK7, OsCPK10, and OsCPK12 act as positive regulators of disease resistance (Mall et al., 2011;Fu et al., 2013). Among the CDPK genes responsive to heat and R. solanacearum inoculation, three CDPK genes (CaCDPK18, -20, and -21) were downregulated by heat stress, and CaCDPK10 was downregulated by R. solanacearum inoculation, indicating that the response of pepper to heat and R. solanacearum inoculation are fine tuned by various CDPK genes, with the majority of them acting as positive regulators and a small number of them acting as negative regulators. Similar observations that numerous CDPK genes were transcriptionally modified by one stress were found in the responses of cotton CDPK genes to cold , of poplar CDPK genes to drought (Zuo et al., 2013), and of rice CDPK genes to cold and salt (Ray et al., 2007). Some CDPK genes in the pepper genome shared similar expression profiles in response to a specific stress, for example, similar expression levels were observed between CaCDPK25, CaCDPK13, and CaCDPK5; CaCDPK30 and CaCDPK23; CaCDPK18, CaCDPK20, and CaCDPK21; CaCDPK1 and CaCDPK4; and CaCDPK31 and CaCDPK17 upon the exposure to heat stress. In contrast, CaCDPK8, CaCDPK26, and CaCDPK31; CaCDPK15 and CaCDPK17; and CaCDPK27 and CaCDPK22 showed similar expression in pepper plants challenged with R. solanacearum inoculation, implying coordinated regulation of a process in plant response to a specific stress by these CDPK genes. Secondly, although the majority of CDPK genes were specifically expressed under treatment with salt, heat, and R. solanacearum inoculation, some CDPK genes were found to respond to more than one stress. For example, CaCDPK23, CaCDPK25, and CaCDPK31 were upregulated by both salt and heat stress, and CaCDPK17 and CaCDPK31 were upregulated by both heat and R. solanacearum inoculation, and CaCDPK31 was upregulated by salt, heat, and R. solanacearum inoculation. In contrast, CaCDPK20 and CaCDPK21 were upregulated by salt, but downregulated by heat stress. These results suggest that crosstalk between pepper responses to salt and heat, to salt and R. solanacearum inoculation, as well as to heat and R. solanacearum inoculation is mediated by CDPK genes. In particular, CaCDPK31 acts in convergence in the response of pepper to salt, heat, and R. solanacearum inoculation. Similar crosstalk between plant responses to biotic and abiotic stresses have been found to be mediated by CDPK genes; for example, PaCDPK1 was induced by cold, wounding, and pathogen challenge (Tsai et al., 2007), TaCPK7 was induced by drought, salt, cold, and hydrogen peroxide (Geng et al., 2011), and OsCPK12 in rice functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance (Asano et al., 2012).
Collectively, the present study identified 31 CDPK genes and five closely related kinase genes in the pepper genome, which can be phylogenetically classified into four distinct subfamilies and are unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures are shared by CDPKs of pepper within the same subfamily, and the expansion of CaCDPK family in pepper was due to segmental duplication events. Five CDPK genes in CM334 were found to be mutated in Chiltepin, and one CDPK present in CM334 was lost in Chiltepin. The majority of the CDPK and CRK genes showed differential expression in different pepper tissues and organs. In addition, 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and R. solanacearum inoculation, respectively; the majority of these genes responded specifically to one stress, whereas a small number of these genes responded synergistically to two or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum inoculation. Our results will prove helpful for the functional characterization of pepper CDPK and its closely related gene families.