Background

Ca²⁺ has been regarded as an important secondary messenger with conservative molecular structure in eukaryotic signal transduction for a long time. Calcium-dependent protein kinases and Ca²⁺ receptors/protein kinase effectors with conservative encoding structure can be found in plants. The role of CDPKs is to perceive the changes of the intracellular Ca²⁺ concentration and convert them into specific phosphorylation events to further activate downstream signal transduction. There have been numerous studies on the role of CDPKs in abiotic stress and immune signaling, while little is known about that in the long-term evolution and development of plants. This study identified the target proteins of CDPKs in plants, including the analysis of phosphorylation sites in target proteins, which might pave the way to elucidate the mechanism for CDPKs at molecular and biochemical level.

The analysis of specific phosphorylation patterns of target proteins indicated that CDPKs acted as signaling centers in the stress signal transduction and development of plants. The family of CDPKs genes included four subfamilies (Harmon et al., 2000; Cheng et al., 2002). 34 family members of CDPKs were identified in Arabidopsis thaliana, 29 family members in Oryza sativa, 20 family members in Triticum aestivum, 35 family members in Zea mays, and 20 family members in Populus trichocarpa (Harmon et al., 2000; Asano et al., 2005; Li et al., 2008; Ma et al., 2013; Zuo et al., 2013). CDPKs had conservative molecular structure, which consisted of a variable N-terminal, a Ser/Thr kinase domain, and a CDPK activation domain (CAD). CAD was composed of pseudo-substrate domain (inhibitory connecting domain) and Ca²⁺ binding domain (also known as calmodulin-like domain) which was highly homologous with calmodulin. Typical calcium binding domain of CDPKs contained up to 4 EF-hand motifs (Harper et al., 2004; Harper and Harmon, 2005; Boudsocq and Sheen, 2013; Liese and Romeis, 2013). Combining Ca²⁺ with CDPK activation domain would induce changes of the whole CDPKs conformation and cause the pseudo-substrate domain to leave the active sites and then kinase activity was activated, making CDPK functionable (Liese and Romeis, 2013).

CDPKs are involved in many aspects of plant life processes, from environment stress perception and signal transduction to hormone regulation at different developmental stages (Cheng et al., 2002; Ludwig et al., 2004; Asano et al., 2012; Boudsocq and Sheen, 2013). CDPKs play a significant role in plant development. The best known example is the process of pollen tube growth. In elongated pollen tube, Ca²⁺ presents a volatile gradient. 12 family members of the 34 members of CDPKs gene are highly expressed in Arabidopsis thaliana pollen (Myers et al., 2009). In the double mutants of AtCPK17/AtCPK34 and AtCPK11/AtCPK24, pollen tube growth were deeply flawed (Myers et al., 2009; Zhao et al., 2013). Another important feature of CDPKs is to participate in Ca²⁺-related signal transduction induced by abiotic stress factors such as salt, drought and low temperature, especially ABA-dependent signal transduction. There have been plenty of reports on changes of CDPKs-dependent ion channels, intracellular metabolic changes, and gene expression changes. CDPKs are considered to have the function of promoting the resistance to abiotic stress, which are able to boost the overexpression of resistance-related kinases in plants as well (Asano et al., 2012; Boudsocq and Sheen, 2013). CDPKs have been acknowledged as a key participator to convert Ca²⁺ concentration changes which is induced by pathogen infection signals into plant defense responses. These include ROS synthesis, gene expression changes, plant hormone synthesis and signal transduction, even apoptosis (Tena et al., 2011; Boudsocq and Sheen, 2013). After pathogen infection, CDPKs were activated rapidly and then showed lower expression after 15-120 min (Romeis et al., 2000), which indicated that CDPKs played a fundamental role in plant immune responses.

Medicago Sativa, as an important pasture, is major feed source for animal husbandry, which is of great significance in economics. Besides, alfalfa cultivars generally have the characteristics of strong resistance and wide adaptability. In this study, through bioinformatics data and analysis technique, we identified family members of the CDPKs gene in two varieties of Medicago, namely Medicago truncatula and Medicagolupulina L. We analyzed the structure, evolution and expression of Medicago CDPKs genes and preliminarily predicted the functions of related genes. This study identified 1 CDPKs gene related to salt-tolerance of Medicago truncatula, 7 CDPKs genes related to nodule growth of Medicago truncatula, and 9 CDPKs genes related to root rot infection of Medicago truncatula, which might lay research basis for further gene cloning and function analysis of these genes.

1 Results and Analysis

1.1 Identification and preliminary analysis of CDPKs genes

Genome sequencing of Medicago truncatula had been completed (JCVI, http://medicago.jcvi.org/medicago/). We took CDPKs family members of Arabidopsis thalianaas as query, and retrieved databases of genome sequences (BLASPn) and genome protein sequences (BLASTp) of Medicago truncatula with local BLAST software. Parameter settings for BLAST used the default values. In addition, we downloaded serine and threonine kinase domain (PF10494) and EF-hand domain (PF00036) from Pfam, and built Hidden Markov Model to search databases of local genomes and protein sequences of Medicago truncatula. Genes with both of the 2 domains were ranked as CDPKs candidate genes of Medicago truncatula for further analysis. As for the genes with multiple expressed sequences on a gene locus, we retained the longest amino acid sequence for further analysis.

We integrated the CDPKs candidate genes of Medicag otruncatula (Table 1) obtained from domain retrieval with HMMER software and local BLAST retrieval, and analyzed the domains on SMART protein domain site. Complete genes both with serine/threonine kinase domain and EF-hand domain were considered to be CDPKs genes of Medicago truncatula. 25 CDPKs genes existed in Medicago truncatula genome. There were no significant differences in size among these 25 CDPKs genes. The smallest Medtr3g098070 was composed of 495 amino-acids and the size of protein was about 55.92 KD, while the largestMedtr1g054865 consisted of 607 amino-acids and the size of protein was 66.45 KD. The isoelectric point distribution of CDPKs genes of Medicago truncatula was relatively centralized as well, and the isoelectric points of 23 genes were less than 7 (acidic). The isoelectric points of proteins corresponding to the only 2 genes, Medtr5g092810 (7.67) and Medtr5g022030 (9.1), were greater than 7. CDPKs gene of Medicago truncatula contains 4 EF-hand domains, except Medtr8g043970, which contains 3.

Medicago lupulina L. is a common species of Medicago in karst areas in Guizhou Province, which has adapted to the special conditions of karst areas with high exchangeable calcium content and arid surface. We completed transcriptome sequencing for tender leaves of Medicago lupulina L., and then conducted retrieval and analysis (mentioned above) to the sequences obtained, found that there were only 4 complete genes with both serine/threonine kinase domain and EF-hand domain. Next we analyzed the genetic relationship among these four CDPKs genes of Medicago lupulina L. (Table 2) and that of Medicago truncatula.

1.2 Phylogenetic relation analysis of CDPKs genes of Medicago

We downloaded CDPKs sequences of Oryza sativa from Phyzome website, together with CDPKs sequences of Arabidopsis thaliana, Medicago truncatula and Medicago lupulina L., and constructed the phylogenetic tree by Neighbor Joining (Figure 1). Amino acid sequences in CDPKs of Arabidopsis thaliana were divided into 4 groups (Cheng et al., 2002). The phylogenetic tree we constructed had this characteristic as well, which was divided into 4 branches. CDPKs sequences of Medicago Sativa were closely related to the gene sequences of Arabidopsis thaliana, which was inconsistent with U-box gene family. U-box gene family of Medicago truncatula was more closely related to the sequences of Oryza sativa but relatively distant from Arabidopsis thaliana (Zheng et al., 2015). One of the genes of Medicago truncatula, Medtr8g043970, together with Os06g50146.1 of Oryza sativa, was both genetically distinct from other CDPKs genes.

1.3 Analysis of CDPKs domains and gene structure of Medicago truncatula

Genome sequence and cDNA sequence of Medicago truncatula exist simultaneously in JCVI, the bioinformatics database of Medicago truncatula. We extracted cDNA and gene sequence of CDPKs gene of Medicago truncatula and analyzed gene structure in GSDS (gene structure display server) (Figure 2; Figure 3). CDPKs genes of Medicago truncatula generally have 7 or 8 exons and the position of introns is more conservative. Except Medtr8g043970, CDPKs gene of Medicago truncatula included 4 EF-hand domains. Medtr8g043970 only contained 3 EF-hand calcium binding domains.

1.4 Chromosome localization analysis of CDPKs in Medicago truncatula

The location information of CDPKs gene of Medicago truncatula could be found in JCVI. We used the location information of CDPKs gene of Medicago truncatula to identify the location of each CDPKs gene on 8 chromosomes of Medicago truncatula by Map Inspect software (Figure 4). CDPKs gene distributed unequally on chromosomes of Medicago truncatula. There was no CDPKs gene on chromosome 2 and 6. Chromosome 1 had a maximum of 6 CDPKs genes. Among them, Medtr1g041150, Medtr1g054865 and Medtr1g055255 aggregated atone place. Besides, phylogenetic analysis showed that these three genes had orthologous relationships, and gene structure analysis indicated that they were genes with conserved structure. Further experiments are needed to analyze that whether Medtr1g041150, Medtr1g054865 and Medtr1g055255 are amplified from the same gene.

1.5 CDPKs gene expression and possible function prediction of Medicago truncatula

We found expression data of 17 genes in 25 CDPKs genes of Medicago truncatula from Medicago truncatula genechips database (http://mtgea.noble.org/v2/). The clustering analysis was processed on the expression data of these genes by Cluster software. CDPKs gene of Medicago truncatula showed obvious gene expression specificity (Figure 5). Expression status of the same gene was very different in roots, stems, leaves, flowers, petioles and leaf buds. A lot of CDPKs genes expressed in roots. Among 17 CDPKs of Medicago truncatula with expression data, 10 of them were detected highly expressed in roots. In contrast, among these 17 CDPKs genes, only Medtr5g089320 expressed in leaves. And it specially expressed in leaves and flowers. During the development of Medicago truncatula root nodules, the expression of CDPKs gene in roots dramatically changed. The expression level of Medtr3g051770 reduced with the growth of root nodules, while that of Medtr7g106710 increased. Besides, during the growth of root nodules, the expression level of Medtr5g009830, Medtr5g089320, Medtr7g068710, Medtr3g098070 and etc. all changed. Not many CDPKs genes in roots involved in the response to salt stress. The Medtr5g022030 didn’t express in roots under control condition. After salt stress, its expression level raised and maintained a high level. By contrast, root rot infection had a powerful influence on gene expression profile of CDPKs in roots of Medicago truncatula. Expression levels of Medtr7g106710, Medtr3g051770 and Medtr8g095440 increased after root rot infection, while expression levels of Medtr7g091890, Medtr4g132040, Medtr1g041150, Medtr3g098070, Medtr8g043970 and Medtr8g099095 decreased.

2 Discussion

Calcium plays an important biological role in plants (cell wall formation, pollen tube development, biotic and abiotic stress signal transduction, etc.). CDPKs genes able to perceive the changes of the intracellular Ca²⁺ concentration and convert them into specific phosphorylation events to further activate downstream signal transduction. CDPKs are intricately involved in many aspects of plant life processes, from environment stress perception and signal transduction to hormone regulation at different developmental stages (Cheng et al., 2002; Ludwig et al., 2004; Asano et al., 2012; Boudsocq and Sheen, 2013). In this study, through bioinformatics data and analysis technique, we identified family members of the CDPKs gene in two varieties of Medicago, namely Medicago truncatula and Medicago lupulina L. We analyzed the structure, evolution and expression of CDPKs genes and preliminarily predicted the functions of related genes.

Medicago lupulina L. is a common species of Medicago in karst areas, which has remarkable ability to adapt to the special conditions of karst areas with high calcium content and arid surface. In this study, we identified 4 complete CDPKs genes from the leaf transcriptome of Medicago lupulina L., which was far less than what it obtained from genome sequences of Medicago truncatula, 25 genes. That should be related to the tissue specific expression of CDPKs genes. We analyzed the expression of Medicago truncatula CDPKs gene and found that only 1 Medicago truncatula CDPK highly expressed in leaves (Medtr5g089320). However, that gene (Medtr5g089320) was not orthologous to the genes found in the transcriptome of Medicago lupulina L.

The analysis of genetic evolution and chromosome localization of 25 CDPKs genes of Medicago truncatula suggested that Medtr1g041150, Medtr1g054865 and Medtr1g055255 aggregated at the same position of chromosome 1. Besides, these 3 genes with conservative genetic structure were orthologous. The amino acid sequences of these three genes showed 97% homology, with only a few different amino acids in the hyper variable region of N-terminal. Further experiments are needed to analyze that whether Medtr1g041150, Medtr1g054865 and Medtr1g055255are amplified from the same gene. It is a pity that we have not found the expression data of these 3 genes, thus it is unable to analyze expression characteristics and gene functions of these 3 genes.

Existing Medicago truncatula gene chip expression data was used to analyze the expression of CDPKs gene of Medicago truncatulain in different organs and under different treatments, and multiple genes related to salt-tolerance, root rot infection and nodule growth of Medicago truncatula were found, which might lay research basis for further gene cloning and function analysis of these genes.

3 Materials and Methods

3.1 Data collection and CDPKs gene excavation

The sequences of Medicago truncatula used in this study were downloaded from Medicago truncatula sequence database (JCVI, http://medicago.jcvi.org/medicago/) (Tang et al., 2014). The transcriptome sequence of Medicago lupulina L. used in this study came from tender leaves of Medicago lupulina L. cultivated out of the soil with high calciumin greenhouse, which was handed over to the sequencing company for transcriptome sequencing. Serine/threonine protein kinase domain (PF10494) and EF-hand domain (PF00036) were downloaded from Pfam (Finn et al., 2006).

The software used for CDPKs gene excavation was local BLAST and HMMER with default arguments for sequence searching. And online software SMART and Expasy were used for the analysis to characteristics of domain and protein of sequences obtained by searching.

3.2 The construction of CDPKs gene phylogenetic tree

CDPKs sequences of Oryza sativa were downloaded from Phyzome. And CDPKs gene sequences of Arabidopsis thaliana were downloaded from Tair. The construction the phylogenetic tree of amino acid sequence in CDPKs gene of Oryza sativa, Arabidopsis thaliana, Medicago truncatula and Medicago lupulina L. employed MEGA 6.0 (Tamura et al., 2013). ClustW method was used for Alignment with default arguments. The phylogenetic tree was constructed by Neighbor Joining.

3.3 Construction of CDPKs genetic map

The location information of CDPKs gene came from bioinformatics database of Medicago truncatula (JCVI, http://medicago.jcvi.org/medicago/) (Tang et al., 2014). Gene positioning map was constructed by MapInspect.

3.4 Analysis of CDPKs gene expression

CDPKs gene expression data of Medicago truncatula was downloaded from Medicago truncatula Gene Expression Atlas of Noble Research Institute (http://mtgea.noble.org/v2/) (He et al., 2009). Cluster analysis of gene chip data used Cluster 3.0. Java Treeview was used for observing clustering results.

Authors’ contributions

GJY was the designer and executor of this study, finishing the paper writing; ZYB completed the data collecting of this study; LF was responsible for furnishing transcriptome data of Medicago lupulina L.; HXH participated in experimental design and paper modification; ZXM was in charge of the experiment, guiding paper writing and modification; YY offered invaluable guidance for paper writing. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the Science Foundation of Guizhou Province (2017GZ66249).

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