Genomics and evolutionary aspect of calcium signaling event in calmodulin and calmodulin-like proteins in plants
© The Author(s). 2017
Received: 26 September 2016
Accepted: 8 January 2017
Published: 3 February 2017
Ca2+ ion is a versatile second messenger that operate in a wide ranges of cellular processes that impact nearly every aspect of life. Ca2+ regulates gene expression and biotic and abiotic stress responses in organisms ranging from unicellular algae to multi-cellular higher plants through the cascades of calcium signaling processes.
In this study, we deciphered the genomics and evolutionary aspects of calcium signaling event of calmodulin (CaM) and calmodulin like- (CML) proteins. We studied the CaM and CML gene family of 41 different species across the plant lineages. Genomic analysis showed that plant encodes more calmodulin like-protein than calmodulins. Further analyses showed, the majority of CMLs were intronless, while CaMs were intron rich. Multiple sequence alignment showed, the EF-hand domain of CaM contains four conserved D-x-D motifs, one in each EF-hand while CMLs contain only one D-x-D-x-D motif in the fourth EF-hand. Phylogenetic analysis revealed that, the CMLs were evolved earlier than CaM and later diversified. Gene expression analysis demonstrated that different CaM and CMLs genes were express differentially in different tissues in a spatio-temporal manner.
In this study we provided in detailed genome-wide identifications and characterization of CaM and CML protein family, phylogenetic relationships, and domain structure. Expression study of CaM and CML genes were conducted in Glycine max and Phaseolus vulgaris. Our study provides a strong foundation for future functional research in CaM and CML gene family in plant kingdom.
KeywordsCalmodulin Calmodulin-like Calcium signaling EF-hands Evolution
In the nuclear fusion of stars and sun, the elements were evolved from hydrogen . During the process of evolution, the element calcium (Ca) was born by successive capture of α particle by oxygen and neon in the process of nuclear fusion [1, 2]. After about 10 billion years, the cell membrane most likely shown its charged activity locally with relentless entropy . To adapt to changing environment, cell must respond to changing environmental signals, and cellular signaling requires an efficient messenger that can move through all parts of the cell to decipher the message. Calcium ion commonly fulfills this signaling role. The concentrations of signaling molecules vary in the cell with time and environmental conditions. The speed and effectiveness of the Ca2+ ion is 20,000 fold higher in the intracellular (~100 nM) compartment than the extracellular (~2 mM) compartment . Cells use a great deal of energy to induce changes in Ca2+ concentration and stabilize the cell. The concentration of Mg2+, which is popularly known as a cousin of Ca2+ doesn’t differ greatly across the cellular compartments. Then question arises, why the concentration of Ca2+ is very less in the cytosol? This is because Mg2+ binds the cytosolic water molecules less efficiently than phosphates. Therefore, if there will be higher Ca2+ concentrations in the cytosol, Ca2+ will bind with phosphate and thus turning the cell into a bone like structure. Unlike other complex molecules, Ca2+ cannot be altered chemically. Therefore, it is necessary to control the cytosolic Ca2+ concentration to avoid any precipitation with the phosphate in the cytosol. Hence, cells have developed necessary cellular mechanisms to control the cytosolic Ca2+ concentration by chelating, compartmentalizing or extruding the ion from the cell. Hence hundreds of proteins have evolved to bind the Ca2+ ion over a million-fold range of affinities (nM to mM) to buffer or lower Ca2+ level in the cell. One of the most important protein chelators of Ca2+ ion is the EF-hand domain containing proteins. There are hundreds of EF-hand containing proteins present in the plants. These proteins are found as family proteins. Some of the important EF-hand domain containing families of proteins are calcium dependent protein kinase (CDPK) [3, 4], calcium dependent protein kinase related kinase (CRK) , calcineurin-B like (CBL) , calmodulin (CaM) and calmodulin like (CML) protein . The CDPK contains the kinase domain, auto-inhibitory domain and a regulatory domain that contains four calcium binding EF-hands while CRK contains kinase domain, auto-inhibitory domain and a regulatory domain that contain only three calcium binding EF-hands. Additionally, the CBL contains only three EF-hands and no kinase domain while CaM and CML contain only four EF-hands and lack a kinase domain [3, 6, 7]. The calcium ion binds to the Asp (D) or Glu (E) amino acids of the EF-hands. The D and E amino acids in the EF-hands are reported to be conserved and present as D-x-D or D/E-E-L motif [8, 9]. The D-x-D motifs are conserved at 14, 15 and 16th position of the EF-hands [8, 9]. Detailed investigations of different genomics and evolutionary aspects of the CDPK and CBL protein family have been discussed recently [8, 9]. However, there have been only little information is available regarding the detail study of CaM and CML gene family in the plants. Therefore in this study, we conducted genome-wide identification of CaM and CML gene family members in plants and analyzed their genomic and evolutionary aspects. Along with the reports of CDPK and CBL protein family, this study completely unveils the genomic aspects of calcium signaling events in plants and calcium signature motifs in EF-hand domains.
Results and discussion
Genomics of CaMs and CMLs
The CaM and CML protein family members of different plant species. A particular protein was considered as either CaM or CML that contained only four calcium binding EF-hands. In total, 41 species were studied to identify CaM and CML protein family. In the table CaM stands for calmodulin and CML stands for calmodulin-like
Gene abbreviation (CaM/CML)
Genome size (Mbs)
Total No. of protein coding genes
No. of CaMs
No. of CMLs
% of CMLs compared to CaMs
Normal distribution of CaMs and CMLs in plant lineage. In the table, P = probability, X = CaMs/CMLs, * = lowest number of CaMs/CMLs in the specified group, ** = highest number of CaMs/CMLs in the specified group
No. of species (N)
0.9345 P (X > 2*)
0.2389 P (X > 6**)
0.7611 P (X < 6**)
0.9756 P (X > 1*)
0.017 P (X > 13**)
0.983 P (X < 13**)
0.7611 P (X > 8*)
0.2389 P (X > 9**)
0.7611 P (X < 9**)
0.7852 P (X > 5*)
0.0778 P (X > 9*)
0.8222 P (X < 9**)
0.9798 P (X > 1*)
0.0256 P (X > 13**)
0.9744 P (X < 13**)
0.9767 P (X > 1*)
0.0113 P (X > 13**)
0.9887 P (X < 13**)
0.7967 P (X > 2*)
0.0344 P (X > 17**)
0.9656 P (X < 17**)
0.9931 P (X > 2*)
0.0015 P (X > 47**)
0.9985 P (X < 47**)
0.7611 P (X > 20*)
0.2389 P (X > 27**)
0.7611 P (X < 27**)
0.8133 P (X > 17*)
0.0202 P (X > 33**)
0.9798 P (X < 33**)
0.975 P (X > 8*)
0.0013 P (X > 47**)
0.9987 P (X < 47**)
0.9738 P (X > 2*)
0.0022 P (X > 47**)
0.9978 P (X < 47**)
Two sample t-test between CMLs and CaMs
Degree of freedom
Degree of freedom
Genome-wide analysis of the CML gene family in plants showed that the green algae C. subellipsoidea, O. lucimarinus, and C. reinhardtii encoded lower numbers of CMLs than the higher plants (Table 1). The genome of C. subellipsoidea and O. lucimarinus encoded only three and two CaMs, respectively. These two species encoded the same number of CMLs, whereas C. reinhardtii encoded six CaMs and three CMLs respectively. C. reinhardtii encoded more CaMs (six) than CMLs (three). Conversely, O. lucimarinus encoded equal numbers of CaMs and CMLs (two). When compared with O. lucimarinus, V. carteri was also found to contain similar numbers of CaMs and CMLs (four) (Table 1). A. thaliana encoded maximum of 47 CML genes while B. rapa encoded 36 CMLs. The tetraploid species G. max and P. virgatum encoded 26 and 20 CMLs respectively. The monocot plant O. sativa encoded 33, while S. bicolor and P. hallii encoded 27 CMLs. On the other hand, P. patens, and S. italica encoded 17 CMLs each. C. clementina, F. vesca, and M. guttatus encoded 19; A. coerulea, C. sativus, L. usitatissimum, P. persica, and Z. mays encoded 21 CMLs each. G. max, S. lycopersicum, S. tuberosum, and T. halophila encoded 27 CMLs each. This distribution of the CML gene family shows, several plant species has encoded the same numbers of CML genes while other do not. The percentage analysis comparison between CaM and CML shows, T. cacao encoded 700% and O. sativa 660% more CMLs compared to their counterpart CaMs (Table 1). The normal distribution study shows, the probability of occurrence of more than two CMLs in a plant genome was 0.9706 (97.06%) while the probability of occurrence of more than 47 CMLs was 0.0024 (0.24%) only (Table 2). The details about the probability of distribution of the CMLs among different groups are mentioned in Table 2. The student’s t-test was conducted to understand the significance of differences between gene numbers present between CaM and CML gene family. Both unpaired and paired t-test analysis shows CaM and CML gene family group members were significantly different from each other (Table 3). These changes in gene family size and unequal distribution of CaMs and CMLs may be attributed to their ploidy level and different cellular processes require for different plants , but they were not related to the size of the genome (Fig. 1). Because in principle, addition or evolution of more genes or genomic content within the genome will lead to increase in the genome size, but vice versa (increase in genome size will lead to more number of genes in a genome) is not true. This might have occurred because of the different cellular and ecological strategies associated with adaptation and expansion of the gene family [10–12]. The variations in the gene family size were largely attributed to the important mechanisms that shape natural variation and adaptation in different species .
CMLs and CaMs Contain varied numbers of introns
Genome-wide analysis of the CML gene family in plants revealed that larger parts of the CMLs were intronless. Among the studied 831 CMLs of 41 species, 596 genes (71.72%) were identified to be intronless (Additional file 2: Table S2) whereas 79 had one intron (9.5%), 24 had two introns (2.88%), 44 had three introns (5.29%), 29 had four introns (3.48%), and 15 had five introns (1.8%). Only a few CMLs contained six, seven, eight or nine introns, and none of them were found to contain ten or more than ten introns (Additional file 2: Table S2). In opposite to CMLs, the majorities of CaMs were contained introns. Among the studied 271 CaMs of 41 species, 14 (5.16%) were found to be intronless, 113 (41.69%) contained one, 35 (12.91%) contained two, 86 (31.73%) contained three, six (2.21%) contained 4, five (1.84%) contained five, and seven (2.58%) contained six introns respectively. The evolutionary perspectives regarding the presence of introns in eukaryotic protein coding genes are not yet clear. However, Mattic  reported that introns can function as a transposable element and nuclear introns has originated from the self splicing group II introns, which later evolved in conjunction with the spliceosome. It assumed that these introns were evolve after divergence from the prokaryotes and later established in the eukaryotic genome with new genetic space and function, which provided a positive pressure for their expansion . According to this concept, it can be speculated that the majority of CMLs were intronless and can therefore be considered older than CaMs. A few CMLs contains introns in their genes, and it is believed that these introns were evolved recently with CaMs. This explains why the intron containing CMLs contains only one (9.5%) intron in their gene. Similarly, a few CaMs were also intronless (5.5%), which indicates that the genome has yet to incorporate the introns into the CaMs. Some other CaMs contains either one (42.44%), two (34.81%) or three (32.22%) introns. This could be possible because these introns were might have added recently and the genome did not got ample time to add more introns into the CaMs. Similarly, the introns present in CMLs are assumed to have been added recently. It requires sufficient time to carry out a major evolutionary event and the addition of more introns into a gene.
According to the intron late hypothesis, introns are the eukaryotic novelty and new introns are emerging continuously during the evolution of eukaryotic genome . Different genes in eukaryotic organisms differ dramatically in terms of density and size distribution. In some cases, zero to six introns per kilobase were observed in the eukaryotic genome [15, 16]. Comparative analysis of exon-intron structures of orthologous genes in higher eukaryotic organisms revealed that they share approximately 25% to 30% of the introns . The presence of 71.72% intronless genes in CMLs shows that the CMLs of plants are highly orthologous and conserved genes in the plant kingdom that evolved from a common ancestor. Similarly, the presence of 42.44%, 34.81% and 32.22% similarity for one, two and three introns containing genes, respectively, shows their close homology with orthologous genes. Intron loss events dominate the short evolutionary distances, whereas intron gain accompanies important evolutionary transitions. Intron gain is an ongoing process, and a high rate of intron gain has been reported for paralogous genes in the model plant Arabidopsis thaliana and Oryza sativa [17–19]. The shared introns were likely derived from a common ancestor of the corresponding species, while the lineage-specific introns were introduced into the genes at the subsequent stages of evolution.
CaM contains four D-x-D motifs and CML contains One D-x-D-x-D motif in their EF-hands
CML contain signal sequences while CaM do not
CMLs were evolved earlier than the CaMs
CaM and CMLs are differentially expressed in different tissues
Tissue specific expression of CaM gene family of Glycine max and Phaseolus vulgaris. All the expression data are presented as FPKM (Fragments per kilobase of exon per million reads mapped)
Tissue specific expression of CML gene family of Glycine max and Phaseolus vulgaris. All the expression data are presented as FPKM (Fragments per kilobase of exon per million reads mapped)
When compared to PvulCaMs, PvulCMLs were also expressed at relatively lower levels. The PvulCML3-3 was ubiquitously expressed in pods (31.86), nodules (39.28), flowers (53.71), stems (87.46), leaves (8), and roots (73.65) (Table 5) while PvulCML3-2 was found to be expressed significantly higher in nodules (72.86), flowers (30.79), stems (36.36), leaves (7.38) and roots (38.76), but expressed to a lesser extent than that of PvulCML3-1. The PvulCML38-3 was highly expressed in pods (74.9) and roots (38.55) followed by expression of PvulCML25-3 in pods (34.2), flowers (96.27), stems (38.21), and roots (38.55) (Table 5). The PvulCML20 was highly expressed in pods (33.01), nodules (35.89), flowers (73.85), stems (48.62), leaves (27.89) and roots (37.04) while PvulCML3-4 was not expressed in pods, nodules, stems, leaves and roots but it was relatively highly expressed in flowers (3.58) (Table 5). Similarly, PvulCML15 was not expressed in pods, nodules, stems, leaves and roots while relatively highly expressed in flowers (4.71). Investigations of the expression of G. max and P. vulgaris CMLs revealed that, CML3 and CML 20 were expressed in all tissues in both the plants, while CML3-4, and CML15 (CML15-2 and CML15-3 in the case of G. max) were not expressed in any of the plants.
The CaM and CML gene family from 41 plant species were studied. Study shows the presence of four calcium binding D-x-D motifs in CaM and one D-x-D-x-D motif in CMLs. The number of family members of CaM and CMLs gene family vary significantly and do not correlate to the genome size of the organism. The evolutionary study shows, CMLs were evolved earlier than CaMs and diversified later. Tissue specific expression of CaM and CML shows, these genes plays important role in development of different tissues in G. max and P. vulgaris.
Identification of CaM and CML gene family
The calmodulin and calmodulin-like genes of Arabidopsis thaliana and Oryza sativa were downloaded from the “Arabidopsis Information Resource” database  and “Rice Genome Annotation Project” respectively . The protein sequences of CaM and CMLs of A. thaliana and O. sativa were used as the query sequences in the publicly available phytozome databases to identify the protein sequences of CaM and CMLs of other plant species using BLASTP program . The CaM and CML genes of Picea abies were downloaded from the spruce genome project . The protein sequences of CaM and CML were used to identify the CaM and CML gene family in other plant species. Overall, 41 plant species were considered during the study (Table 1). The statistical parameters used during BLASTP searches were target type, proteome; expect (E) threshold, (−1); and comparison matrix, BLOSUM62. Sequences recovered from the BLASTP searches were collected for further analysis. Later, all the collected sequences of BLAST results were evaluated using the scanprosite software to confirm the presence of the prosite calcium binding EF-hands domain. The sequences those showed the presence of four calcium binding EF-hands domains were considered as CaM or CML proteins. Later, all sequences were subjected to the BLASTP analysis in the A. thaliana (TAIR) and O. sativa proteome (rice genome annotation project) database. The sequences that resulted in BLASTP hits of the CaM gene in both the database were considered as CaM protein while that resulted in BLASTP hits to the CMLs were considered as CML proteins.
Subsequently we named all the CaM and CML proteins of the studied plant species. Nomenclature was conducted according to the orthologous based nomenclature system as proposed earlier [8, 25]. Name were given by taking the first letter of the genus name in upper case and the first letter of the species (sometimes 2 to3 letters were used when redundancy was observed) name in the lower case followed by the number corresponding to the orthologs genes of A. thaliana or O. sativa. Monocot plant species were named according to the orthologous genes of O. sativa while dicot and other species were named according to the orthologous genes of A. thaliana as proposed earlier [8, 25, 26].
Molecular modeling of CaM and CML
Molecular modeling was conducted to evaluate the molecular details of CaM and CML proteins. The Geno3d software  was used to construct the molecular structure of CaM and CMLs. The protein sequence of AtCaM1 and AtCML1 was utilized as the query sequence to search the model. Following statistical parameters were used to run the analysis: database, non-redundant protein sequences; filter query sequence (−F), true; expectation value (−e, real), 10.0; number of on-line descriptions (−v, int), 500; number of alignments to show (−b, int), 500; matrix (−M), BLOSUM62; expectation value threshold for inclusion in multipass model (−h, real), 0.002; maximum number of passes to use in multipass version (−j, int), 3.
Multiple sequence alignment
Multiple sequence alignment of CaM and CML proteins was conducted separately to investigate the presence of conserved domains and motifs. Multalin software was used to run the multiple sequence alignment. The statistical parameters used during multiple sequence alignments were, sequence input format, Multalin-fasta; protein weight matrix, BLSOUM62-12-2; gap penalty at opening, default; gap penalty at extension, default; gap penalties at extremities, none; one iteration only, no; high consensus level, 90%; low consensus level 50%.
Palmitoylation site prediction
The palmitoylation sites of CaMs and CMLs protein were predicted using the CSS palm software version 2.0 . During the prediction, input sequences were submitted in FASTA format and the threshold was set to higher or medium.
The phylogenetic trees were constructed to understand the evolution of CaM and CMLs. To construct the phylogenetic tree of CaMs and CMLs, protein sequences were subjected to clustalW or clustal omega software to generate a clustal file . The generated clustal files were then converted to MEGA file format using the MEGA6 software . The generated MEGA files of CaMs and CMLs were used to construct the phylogenetic trees. Different statistical parameters used to construct the phylogenetic tree were as follows: analysis, phylogeny reconstruction; statistical method, maximum likelihood; test of phylogeny, bootstrap method; no. of bootstrap replicates, 1000; substitution type, amino acid; model/method, Jones-Taylor-Thornton (JTT) model; gaps/missing data treatment, partial deletion; site coverage cutoff (%), 95; ML heuristic method, nearest-neighbor-interchange (NNI); and branch swap filter, very strong. The gamma parameter for site rates was estimated using MEGA 6 software. Following parameters were used to study the site rate: analysis, estimate rate variation among sites (ML); statistical method, maximum likelihood; substitution type, amino acid; model/method, Jones-Taylor-Thornton (JTT); rates among sites, gamma distributed (G); number of discrete gamma categories, 5; gaps/missing data treatment, partial deletion; site coverage cutoff (%), very strong. The species tree was built using NCBI taxonomy browser (http://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi).
Tissue specific expression of CaMs and CMLs
Understanding the tissue specific expression of any particular gene is important to elucidating its role in growth, development and stress responses. Therefore, we studied the tissue specific expression of CaM and CML genes of G. max and P. vulgaris. The expression profiles of CaMs and CMLs were searched in the phytomine database of phytozome. The expression profiles of all of the genes are represented as FPK (fragments per kilo base of exon per million reads mapped).
Regression analysis was conducted to evaluate the correlation of CaM and CML gene family size with regard to the genome size. Mathportal (http://www.mathportal.org/calculators/statistics-calculator/correlation-and-regression-calculator.php) was used for the correlation regression analyses.
Calcium dependent protein kinase
CDPK Related kinase
This work was carried out with the support of the Next-Generation Biogreen 21 Program (PJ011113), Rural Development Administration, Korea.
This work was carried out with the support of the Next-Generation Biogreen 21 Program (PJ011113), Rural Development Administration, Korea. The funding agency has no roles in the design of the study and collection, analysis, and interpretation of data.
Availability of data and materials
All data analyzed during this study were taken from publicly available phytozome database and also provided as Additional files.
TKM Conceived the idea, design the experiment, analyzed data and drafted the manuscript; PK and HB Revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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- Clapham DE. Calcium signaling. Cell. 2007;131:1047–58.View ArticlePubMedGoogle Scholar
- Cameron AGW. Nuclear Reactions in Stars and Nucleogenesis. Publications of the Astronomical Society of the Pacific . 1957;69:201.
- Kanchiswamy CN, Mohanta TK, Capuzzo A, Occhipinti A, Verrillo F, Maffei ME, et al. Differential expression of CPKs and cytosolic Ca2+ variation in resistant and susceptible apple cultivars (Malus x domestica) in response to the pathogen Erwinia amylovora and mechanical wounding. BMC Genomics. 2013;14:760.View ArticlePubMedPubMed CentralGoogle Scholar
- Mohanta TK, Sinha AK. Role of Calcium-Dependent Protein Kinases during Abiotic Stress Tolerance. In: Tuteja N, Gill S, editors. Abiotic Stress Response Plants. 2015th ed. byWiley-VCH Verlag GmbH & Co.; 2015. p. 185–208.
- Mohanta TK, Mohanta N, Mohanta YK, Parida P, Bae H. Genome-wide identification of Calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events. BMC Plant Biol. 2015;15:189.
- McCormack E, Braam J. Calmodulins and related potential calcium sensors of Arabidopsis. New Phytol. 2003;159:585–98.View ArticleGoogle Scholar
- McCormack E, Tsai YC, Braam J. Handling calcium signaling: arabidopsis CaMs and CMLs. Trends Plant Sci. 2005;10:383–9.View ArticlePubMedGoogle Scholar
- Mohanta TK, Mohanta N, Mohanta YK, Bae H. Genome-wide identification of calcium dependent protein kinase gene family in plant lineage shows presence of novel D-x-D and D-E-L motifs in EF-hand domain. Front Plant Sci. 2015;6:1146.View ArticlePubMedPubMed CentralGoogle Scholar
- Mohanta TK, Mohanta N, Mohanta YK, Parida P, Bae H. Genome-wide identification of Calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events. BMC Plant Bol. 2015;15:189.View ArticleGoogle Scholar
- Konstantinidis KT, Tiedje JM. Trends between gene content and genome size in prokaryotic species with larger genomes. Proc Natl Acad Sci U S A. 2004;101:3160–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Fischer I, Dainat J, Ranwez V, Glémin S, Dufayard J-F, Chantret N. Impact of recurrent gene duplication on adaptation of plant genomes. BMC Plant Biol. 2014;14:151.View ArticlePubMedPubMed CentralGoogle Scholar
- Lespinet O, Wolf YI, Koonin EV, Aravind L. The role of lineage-specific gene family expansion in the evolution of eukaryotes. Genome Res. 2002;12:1048–59.View ArticlePubMedPubMed CentralGoogle Scholar
- Guo YL. Gene family evolution in green plants with emphasis on the origination and evolution of Arabidopsis thaliana genes. Plant J. 2013;73:941–51.View ArticlePubMedGoogle Scholar
- Mattick J. Introns: evolution and function. Curr Opin Genet Dev. 1994;4:823–31.View ArticlePubMedGoogle Scholar
- Rogozin IB, Carmel L, Csuros M, Koonin EV. Origin and evolution of spliceosomal introns. Biol Direct. 2012;7:11.View ArticlePubMedPubMed CentralGoogle Scholar
- Carmel L, Wolf YI, Rogozin IB, Koonin EV. Three distinct modes of intron dynamics in the evolution of eukaryotes Three distinct modes of intron dynamics in the evolution of eukaryotes. 2007. p. 1034–44.Google Scholar
- Lin H, Zhu W, Silva JC, Gu X, Buell CR. Intron gain and loss in segmentally duplicated genes in rice. Genome Biol. 2006;7:R41.View ArticlePubMedPubMed CentralGoogle Scholar
- Hartung F, Blattner FR, Puchta H. Intron gain and loss in the evolution of the conserved eukaryotic recombination machinery. Nucleic Acids Res. 2002;30:5175–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Babenko VN, Rogozin IB, Mekhedov SL, Koonin EV. Prevalence of intron gain over intron los in the evolution of paralogous gene families. Nucleic Acids Res. 2004;32:3724–33.View ArticlePubMedPubMed CentralGoogle Scholar
- Martín ML, Busconi L. A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature. Plant Physiol. 2001;125:1442–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 2012;40:D1202–10.View ArticlePubMedGoogle Scholar
- Ouyang S, Zhu W, Hamilton J, Lin H, Campbell M, Childs K, et al. The TIGR Rice Genome Annotation Resource: improvements and new features. Nucleic Acids Res. 2007;35:D883–7.View ArticlePubMedGoogle Scholar
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.View ArticlePubMedPubMed CentralGoogle Scholar
- Nystedt B, Street NR, Wetterbom A, Zuccolo A, Lin Y-C, Scofield DG, et al. The Norway spruce genome sequence and conifer genome evolution. Nature. 2013;497:579–84.View ArticlePubMedGoogle Scholar
- Mohanta TK, Arora PK, Mohanta N, Parida P, Bae H. Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants. BMC Genomics. 2015;16:58.View ArticlePubMedPubMed CentralGoogle Scholar
- Hamel L-P, Nicole M-C, Sritubtim S, Morency M-J, Ellis M, Ehlting J, et al. Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 2006;11:192–8.View ArticlePubMedGoogle Scholar
- Combet C, Jambon M, Del G, Geourjon C. Geno3D : automatic comparative molecular. Bioinformatics. 2002;18:213–4.View ArticlePubMedGoogle Scholar
- Ren J, Wen L, Gao X, Jin C, Xue Y, Yao X. CSS-Palm 2.0: an updated software for palmitoylation sites prediction. Protein Eng Des Sel. 2008;21:639–44.View ArticlePubMedPubMed CentralGoogle Scholar
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539.View ArticlePubMedPubMed CentralGoogle Scholar
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.View ArticlePubMedPubMed CentralGoogle Scholar