Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants

Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.


Background
During evolution, plants have developed complex arrays of defense mechanisms to mitigate the copious, often adverse and ever changing environmental conditions. Perception of variations in environmental as well as internal developmental cues, transduction and amplification of signals and activation of the response to stimuli is crucial for survival, optimal growth and development. Protein kinases are important signaling molecules that perceive various signals and transduce them for active responses. These compounds carryout diverse phosphorylation processes at the transcriptional, translational and post-translational level by catalyzing the addition of phosphate groups to serine and threonine/tyrosine residues in their target proteins in both prokaryotic and eukaryotic cells [1,2]. These modifications have led to changes in catalytic activity, affinity and interaction activity of target protein. However, the phosphorylation events in proteins are reversible due to protein phosphatase, enabling maintenance of the balance between kinase driven phosphorylation and phosphatase driven dephosphorylation events [3].
Plant genomes are rich in genes that encodes protein kinases and constitute the kinase super-family [4]. These super families are divided into different classes based on amino acid sequence similarity and functional characteristics. The mitogen activated protein kinase gene family, which is one such family, is known for evolutionary conservation across eukaryotic taxonomic groups and functioning within hierarchical cascades [1]. Phosphorylated proteins carry out a wide array of cellular responses, including changes in gene expression, innate immunity, developmental programmes and stress and hormonal responses [5][6][7][8].
Mitogen activated protein kinases consist of three kinasemodules composed of mitogen activated protein kinase kinase kinase (MAPKKKs), mitogen activated protein kinase kinase (MAPKKs) and mitogen activated protein kinase (MAPKs). In the general model, extracellular signals activate MAPKKKs, which phosphorylate downstream MAPKKs. The phosphorylated MAPKKs in turn phosphorylate MAPKs [9,10]. Protein phosphorylation events may occur throughout the protein kinase sequences, but usually occur on the activation loop [11]. The activation loop, which is present at the C-terminal end, resides within sub-domain VII and VIII of sub-domain eleven [4]. The activation loop contains conserved serine, threonine and/or tyrosine amino acid residues that may be reversibly phosphorylated [6] via cis auto-phosphorylation or trans phosphorylation by upstream kinases [12].
The initial descriptions of components of the MAPK cascade have been provided for the popular model plant, Arabidopsis. Advancements in sequencing technologies and bioinformatics tools have greatly increased the pace of genome sequencing projects, resulting in successful sequencing of several plant genomes. Post genome sequencing projects have enabled relatively easy identification of particular gene families based on conserved signature motifs and sequence similarity. Available genome sequences from several plants genomes have provided us with an opportunity to identify MAPK family members across photosynthetic eukaryotes (plants and algae) that will shed more light on MAPK evolution and signaling in plants and lower photosynthetic eukaryotes. In recent years, identification of MAPK gene family members in plants has been limited to a few species including Arabidopsis thaliana [13], Oryza sativa [14], maize [15], Brassica napus [16], apple [17], and Brachypodium [18]. Further, a study by Janitza et al. [19] and a review article by Doczi et al. [20] have provided a comprehensive overview of the evolutionary history of MAPKs in green plants by using a limited number of plants species.
However, there is currently limited information regarding the nomenclature, conserved structures, genomics and biochemistry of MAPKs in plants. In this communication, we identify the MAPK gene families of 40 different plant species and provide a unique nomenclature to all MAPKs. This nomenclature system can be further applied to newly identified MAPKs of other species. Furthermore, the genomics, biochemistry and conserved consensuses of plant MAPKs describe several novel aspects of plant MAPKs.

Identification and nomenclature of MAPKs
We identified the MAPK gene family from 40 different plant species starting from the unicellular lower eukaryote Chlamydomonas reinhardtii to the multi-cellular angiosperm Arabidopsis thaliana and attempted to cover the maximum number of species across the plant lineage. We found that MAPKs members of a genome varied from species to species across the whole plant lineage. The 40 species collectively gave rise to 589 MAPK sequences. The tetraploid Glycine max contained the most MAPK genes in its genome (31), whereas the lower eukaryotic plant Ostreocccus lucimarinus contained three (Table 1). In addition, Brassica compestris (30), Gossipium raimonddi (28), Malus domestica (28), Panicum virgatum (27), Linum usitatissimum (24) and Populus trichocarpa (21) contained higher number of MAPKs (Table 1, Additional file 1). All the identified 589 MAPKs were provided with specific names according to the orthologous sequence similarity with Arabidopsis thaliana or Oryza sativa.
The molecular weights of MAPK proteins were vary from 22.381 (VvMPK1) to 98.915 (MdMPK20-2) kDa and the isoelectric points vary from 5.00 (MdMPK20-1) to 9.52 (CsubMPK15) (Additional file 2). The isoelectric point (pI) of group A and group B MAPKs were ranges from acidic to slightly acidic, while those of group C and group D were reside within the basic pI ranges. The average amino acid composition of MAPK protein showed that, abundance of leucine (9.63) amino acid was maximum and tryptophan (0.70) amino acid was minimum   Figure 1A, Table 3).

Conserved sequences in activation loop region
As discussed earlier, MAPKs contain the classic T-E-Y or T-D-Y motif in the activation loop region, and we found that majority of MAPK members contain classic  Table 2).

C-terminal conserved sequences
The  Table 3).

Common docking domains
During mitogen activated protein kinase (MAPK) signaling, the ability of MAP2Ks to recognize their cognate From this table it is evident that, the number of genes in a specified gene family don't directly proportional to its genome size.  I  I  I  I  I  I IV  MAPKs are facilitated by presence of short docking motif (D-site) that binds to its target complementary region on the MAPK. Similarly MAPKs are also contain short docking site that recognizes many downstream target proteins by utilizing the same strategy. From the studied MAPKs, we did not find presence of any unique and specific conserved docking domains for all groups of the MAPKs. Instead, conservation of the docking domains consensus is somewhat group specific ( Table 4). The con- Table 4).

Phylogeny
An unrooted phylogenetic tree was constructed to infer group specific relationships of MAPKs. Upon phylogenetic analysis, all studied MAPK genes are fell into six different clusters, that are named according to the MAPK grouping of A. thaliana.  Table 5). The average overall phylogenetic mean distance of plant MAPK is 0.54 (standard error 0.029). During phylogenetic distance estimation, all the positions with less than 95% site coverage are eliminated. That is, fewer than 5% alignment gaps. The missing data and ambiguous bases are allowed at any position.

Statistical analysis
Different statistical analysis was carried out to infer the statistical significance of the study. In Tajima's relative rate test, different MAPK sequences were taken randomly from active data as different groups. Analysis was repeated for two times by taking random MAPK sequences into different taxonomic group A, B and C (these groups are statistical groups and should not be confused with MAPK groups). More specifically, group C was used as out group. When we took MgMPK4-1 (group A), GmMPK16-3 (group B) and AtPIN1 (group C), resulted p-value was 0.05935 and X 2 -test result was 3.56 (Table 6). When MgMPK4-1, GmMPK16-3 and AtCBL1 are taken as group A, B and C respectively, the p-value result was 0.00468 and X 2 -test result was 8.00. In both the cases statistical value was found to be significant ( Table 6). The P-value less than 0.05 is often used to reject the null hypothesis of equal rates between lineages (p ≤ 0.01: very strong presumption against null hypothesis, 0.01 < p ≤ 0.05 strong presumption against null hypothesis, 0.05 < p ≤ 0.1 low presumption against null hypothesis, p > 0.1 no presumption against null hypothesis). The analysis involved 3 amino acid sequences. All positions containing gaps and missing data were eliminated. In Tajima's test (See figure on previous page.) Figure 1 Multiple sequence alignment of plant MAPKs. A. The figure shows presence of N-terminal TEY, TDY, SEY and SDY motifs (in red). The SEY and SDY motifs are aligned with TEY and TDY motifs and very specific to group D MAP kinase genes. Activation loop TDY motifs are marked in blue and indicated inside the box. Conserved signature consensus sequences present within MAP kinases domain of MAPK proteins are marked in maroon. C-terminal conserved motifs are marked in green and presented inside box. All eleven sub-domains are also indicated in the figure. B. Molecular structure of AtMPK1 (group C) and SiMPK20-1 (group D). In AtMPK1, the arrow mark shows presence of TEY motif in the activation loop region. Similarly, in the group D MAP kinase (SiMPK20-1), the conserved N-terminal TEY motif and activation loop TDY motifs are indicated by different arrows.
statistics (D) for neutrality, the D value was found to be 4.904140 (D = 4.904140) ( Table 7). All the positions with less than 95% site coverage are eliminated during Tajima's test for neutrality. There were a total of 322 positions in the final dataset.

Gene duplication
Chromosomes are evolved via fusion, fission, insertion, and duplication events, allowing evolution of chromosome size and number, and hence the genes. Gene duplication is the major force acting on the evolution of different species, and the gene families are groups of genes generated by duplication.  (27).

MAPKs in dicotyledonous plants
The MAPK gene family of dicotyledonous plants has shown large variations among MAPK gene family The N-terminal conserved motifs of plant MAPKs. These motifs are immediately followed by presence of N-terminal SEY, SDY, TEY, TDY motifs of group D MAPKs. This picture is a pictorial representation of different motifs taken from different groups of MAPKs. For details, please see Table 2 and Additional file 4.

MAPKs in lower photosynthetic eukaryotes (Algae, Moss and Pteridophyte)
The lower photosynthetic groups includes four algae, one bryophyte, one pteridophyte and one gymnosperm species (Table 1). Multiple MAPKs were seen in several species of algae (Table 1). Our study also showed multiple MAPKs (TEY, 2/3/4 and TDY, 1/2) in unicellular and multi-cellular algae (

Nomenclature and identification of MAPKs
It is very important to assign an appropriate and specific name to each member of the family to enable a thorough understanding of it. Therefore, we provided unique names to all 589 identified MAPKs across the plant lineage using the orthologous based nomenclature system proposed by Hamel et al. [21]. In the traditional naming system, names are assigned to gene(s) that are identified and cloned first, regardless of their similarities to other gene(s). For example, if someone cloned the ThMPK gene from Thelluginella halophila first, it named as ThMPK1, regardless of its orthologous similarity with other MPKs. Accordingly, if this ThMPK1 has orthologous similarity with AtMPK6, it should be named ThMPK6, but this does not happen. However, orthology lends the legitimacy to transfer of functional similarities from its ancestors [22][23][24]. As a result, orthology based nomenclature can provide succinct information regarding its orthologous counterpart gene. Practically, it is difficult to study every individual MAPK gene in all plant species to understand their specific roles in different  aspects of plant biology. As orthology lends the legitimacy of common ancestry and evolutionary function, orthology based nomenclature will provide ideas regarding possible roles of specific genes in the plant species being investigated. This system of nomenclature can be further extended to newly identified gene families of other plant species.
To date, MAPK gene family members of only few plant species have been reported, including Oryza sativa [14], Arabidopsis thaliana [13], Zea mays [15], Brachypodium distachyon [18], Canola (Brassica napus) [16] and Malus domestica [17]. Although MAPKs from these plant species are previously identified, we have included them here to broaden the study. Inclusion of these species in our study led to identification of some new members of MAPKs. An earlier study by Hamel

Genomics of MAPKs
All species are confined by specific numbers of fundamental traits known as chromosome [25,26]. The number of sets of chromosomes varies among genera and species, as well as within species. Some species are functionally haploid (e.g., Chlamydomonas, Volvox, Coccomyxa, Ostreococcus, Physcomitrella, Selaginella), or diploid (Oryza sativa, Arabidopsis thaliana, Gossipium raimondii, Glycine max, Zea maize) ( Table 1). The genome size of a specific species is directly correlated with the ploidy level (haploid, diploid or polyploidy) of the organism [27,28]. The lower eukaryotic organisms such as Chlamydomonas, Volvox, Coccomyxa, Ostreococcus, Physcomitrella, and Selaginella are very simple life forms relative to higher eukaryotic angiosperms; therefore, they encode very few MAPK genes relative to higher plants. For example, the lower eukaryotic algae O. lucimarinus encodes only three MAPKs, while C. subellipsoidea encodes only four. C. reinhardtii and S. moellendorffii encode six MAPKs each, whereas P. patens encodes eight. The plants G. max has tetraploid genome; hence, its genome size is larger than those of O. sativa and A. thaliana. Owing to the ploidy nature of the genome, organisms encode more MAPKs [27,29,30]. G. max encodes maximum of 31 MAPKs, whereas B. rapa encodes for 30 MAPKs. Similarly, M. domestica and G. raimondii encodes for 28 MAPKs each. The presence of higher numbers of MAPK genes in these plants is attributed to their bigger genome size and ploidy level or whole genome duplication. The number of MAPK genes within each family varies from species to species depending upon the complexity and ploidy level of plants (Table 1).
During this study, we found that MAPK genes harbored several introns (Additional file 1). Additionally, different genes contained different numbers of introns ranging from zero (intronless) to fourteen. We did not find any group specific conserved intron organization for groups A, B, C, and E MAPK genes. However, group D MAPKs harbored seven to fourteen introns. It has been reported that intron organization is conserved at levels up to 10-20% between orthologs [31,32], and the presence of 126 MAPK genes containing nine introns in their gene constitutes 21.39% orthology. Accordingly, our findings are in agreement with earlier findings regarding the orthology based evolution of introns in plants. These findings confirm that group D MAPKs are evolutionarily more conserved than other groups of MAPKs.

Conserved motifs and domains
Mitogen activated protein kinases are multigene families characterized by the presence of an activation loop T-E-Y/ T-D-Y motif. [6,21] These conserved motifs are target phosphorylation sites of upstream MAPKK (mitogen activated protein kinase kinase).To date, there have been no reports regarding the presence of N-terminal conserved motifs in MAPKs. We found that group D MAPKs contains conserved N-terminal T-E-Y/T-D-Y/S-D-Y and S-E-Y motifs ( Figure 1A, 1B, Table 2). Chlamydomonas and Volvox share a common A-V-H motif instead of the S-E-Y/S-D-Y/T-E-Y and T-D-Y motif. Protein phosphorylation can occur on multiple distinct sites throughput the given protein [33,34]. Hence the presences of N-terminal conserved motifs are may be target phosphorylation sites of some other unknown kinases. In addition to the presence of these conserved motifs, the N-terminal region of MAPKs also contains several other conserved consensus amino acids, I-G-x-G-x-Y-G-x-V, I-K-K-I-  Table 3). These conserved consensus amino acid sequences may be considered as signature consensus of MAPKs. The presence of conserved amino acids consensuses in MAPKs reflects their common evolutionary ancestry. Earlier, Hanks (2003) reported that the protein kinase super-family contains the G-x-G-x-x-G conserved consensus sequence [4], which is similar to the results of our study, in which I-G-x-G-x-Y- Table 4 Table showing  G-x-V was conserved instead of G-x-G-x-x-G across all MAPKs ( Figure 1A, Table 3) Table 2). None of the identified motifs belonged to group C MAPK. These new motifs are assumed to be undergoing recent evolution to expand the diversity of MAPKs; however, the absence of a new kind of activation loop motif for group C MAPK indicates that this group is more conserved than other groups of MAPKs. The presence of diverse activation loop motifs are may be for target phosphorylation sites for diverse kinases that can facilitate phosphorylation events more easily to overcome selective pressure. These activation loop conserved motifs are closely followed by several other group specific conserved motifs, including    as other motifs in close vicinity to the activation loop indicates that more in depth investigations are needed to confirm their functions. The presence of group specific conserved motifs and domains explains the orthology based evolution of MAPKs from the common ancestors. In fungi, MAPKs regulates five different pathways namely CWI (cell wall integrity), HOG (high osmolarity glycerol), Kss, Fus, and Smk1 (sporulation and meiosis) [35]. The CWI pathway is carried out by fungal Mpk1 that contain T-E-Y motif in its activation loop domain, HOG pathway is carried out by Hog1 that contain T-G-Y motif, Kss pathway is responsible for filamentous growth in fungi and Kss1 contain T-E-Y motif, Fus pathway is responsible for mating and cell cycle arrest and contain T-E-Y motif in its activation loop domain. The fungal Smk1 pathway responsible for meiosis and sporulation contain T-N-Y motif in its activation loop domain. From these report, it is evident that MAPK that contain different activation loop motif controls different pathways. Presence of several new activation loop motifs in plants may be responsible for some novel pathways which are yet to be elucidated.

Common docking site
It was previously assumed that substrate specificity of an enzyme is determined by stereo-chemical complementarities with its active site [36][37][38][39]; however, these preferences are not stringent enough. It has since been found that, in addition to substrate target site preferences, many protein kinases uses dedicated modular protein-protein interaction docking domains, interactions involving binding of the surface of the catalytic domain, but distinct from catalytic active sites [40][41][42]. These docking grooves are bind to the short peptide docking motifs that are separated from the substrate motif. Although these two supplemental recognition strategies are not mutually exclusive, development of these alternative modes of recognition provides a very simple method to meet increasing evolutionary requirements [41]. Protein kinases often recognize their substrates and regulators through docking interaction sites that occurs outside the active site [43,44], and these interactions can help us to understand the kinase networks. The MAPKs contains group specific conserved docking domains, K-M-L-T-F- Table 4). The presence of group specific docking domains in MAPKs suggests that different MAPKs targets are group specific. Similarly, the presence of group specific conserved docking domains suggests, evolution of MAPKs are orthologous based and are group specific.
In all the four cases, statistical result was found to be significant. The analysis involved 589 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 325 positions in the final dataset. Evolutionary analyses were conducted in MEGA6. Abbreviations: m = number of sequences, n = total number of sites, S = Number of segregating sites, p s = S/n, Θ = p s /a 1 , π = nucleotide diversity, and D is the Tajima test statistic.
sequences. The analysis revealed that all MAPKs fell into six different groups with well supported bootstrap values. We named them as group A (red), B (blue), C (pink), D (purple), E (teal) and F (green) ( Figure 5, Additional file 5). The clustering of MAPKs into different groups reflects their orthology based origin from common ancestors. The cluster of group E MAPKs belonged to lower eukaryotic organisms and fell in the distal end (basal part) of the phylogenetic tree ( Figure 5, Additional file 5). The finding of group E MAPKs from lower green algae and other organisms fell as an independent group suggests that these MAPKs were evolved independently and diverged during evolution to higher eukaryotes. Independent grouping of group E and F MAPKs may also indicate lower eukaryotic specific functions and their independent evolution [45][46][47].
Although biological functions of all MAPKs are not yet understood, MAPKs of the same subgroup are likely to be involved in similar physiological responses, and hence similar functions. The presence of lower eukaryotic specific group F MAPKs in the middle of the phylogenetic tree may also reflects their roles in evolution of MAPKs by subsequent divergence and duplication. During speciation events, these MAPKs became diversified and evolved as multigene families from common ancestors. It has been reported that multiple members of specific gene families of a particular organism are the natural products generated from the evolutionary history experienced by an organism [48][49][50][51]. Accordingly, the presence of several members in a gene family reflects the succession of genomic rearrangement, and its expansion is due to extensive duplication and diversification that occurs during the course of evolution [52,53]. Expansion of the gene family may be involved in different developmental processes of plants [54][55][56][57][58].

Statistical analysis
In the random sampling of MAPK sequences in Tajima's relative rate test, we found significant p-value and X 2test result in both the studied groups. This implies that our study is statistically significant ( Table 6). In Tajima's test for neutrality, the Tajima's D test result is 4.904140 (D = 4.904140) ( Table 7). In Tajima's D test, when D = 0, the average heterozygosity became equal to number of segregating sites and can be interpreted as expected variation is similar to observed variation [59,60]. The evolving population can be due to mutation-drift equilibrium and no evidence of selection. When D < 0, the average heterozygosity is lower than number of segregating sites [59,60]. In this case, it can be interpreted as rare alleles are present at very low frequencies and recent selective sweep led to population expansion after recent bottleneck that contain the linkage to swept a gene. When D > 0, the average heterozygosity is more than that of segregating sites and can be considered as presence of multiple alleles; some at low and others at high frequencies [59,60]. This creates balancing in selection by sudden contraction in population. Tajima's negative D value signifies excess low frequency of polymorphism relative to expectation. This indicates expansion in a population size by selective sweep or by purifying selection. Tajima's positive D value signifies high frequencies of polymorphism indicating decrease in a population size by balancing selection. Tajima's D value greater than +2 or less than −2 are considered as significant [59,60]. In our result we found D value of 4.904140 which is greater than 0 (D > 0) and +2 (Table 7). This signifies MAPKs are undergone high frequencies of polymorphism by decreasing population size due to balanced selection. So, the heterozygosity of plant MAPKs are more than that of number of segregating sites and presented as multiple alleles.

Duplication of MAPKs
The size of the plant genome and number of chromosomes in each genome varies widely among species and shows diversity of 2350-folds ranges from 63 to 149,000 Mb that divided into n = 2 to n =~600 chromosomes [25]. Chromosomes evolved by fusion, fission, insertion, and duplication events, allowing evolution of chromosome size and number and hence the genes [61,62]. Vascular plants evolved approximately 410 million years ago and diverged into several lineages, among which lycophytes, ferns and gymnosperm and seed plant survived [63]. The transitions from aquatic to land plants, gametophytic generation to sporophytic generation, non-vascular form to vascular form and non seed bearing to seed bearing life cycles requires evolution of new genes [63,64]. The co-linearity resulting from the common ancestry of angiosperms provides a powerful method to determine the orthology [65][66][67]. The MAPK genes from diverse species investigated in this study were found to be orthologous. Based on the results of this study, the orthologous MAPK genes have undergone duplications and given rise to several paralogous genes. The MAPKs those contained novel activation loop motif were found to be duplicated ( Table 8). The most common MAPK (AtMPK1) that contain classical T-E-Y motif in its activation loop motif is highly duplicated with z-score 19.46 and 100% confidence level. The AtMPK20 that contained T-D-Y motif in its activation loop motif resulted in z-score of 11.88 with 100% confidence interval. All the newly identified MAPKs that contained novel activation loop motifs resulted in z-score more than four with 100% confidence level. Genes with zscore value four or more than four were considered as highly significant to be duplicated [68]. The phylogenetic study showed that MAPK genes of the grass family of monocotyledonous plants (O. sativa, S. bicolor, B. distachyon, P. virgatum, S. italica and Z. mays) are more conserved and clustered together, demonstrating that grass family MAPKs were evolved from a single lineage. It has been reported that, although genomes of the grass family differ greatly in terms of size, ploidy level and chromosome number, their genetic markers and genes are very well conserved between genomes [69,70]. It has also been reported that there are several duplicated MAPK genes in grass family plants due to their ploidy level and genome duplication [71,72]. Vascular plants appeared approximately 410 million years ago, then diverged into several lineages [73]. The first non-seed vascular plant, Selaginella, lacks evidence of whole genome duplication or polyploidy, which explain why S. moellendorffii contains only six MAPK genes. Although the genome sizes of S. moellendorffii and A. thaliana are very similar [63], A. thalaiana contains 16,574 (65%) duplicated genes [62]. The transition from a gametophyte to sporophyte dominant life cycle requires far fewer new genes than the transition from non-vascular non-flowering to vascular and flowering plants [63]. Earlier studies of Chlamydomonas, Physcomitrella, Selaginella and fifteen other angiosperm species by Banks et al., revealed that transition from single celled green algae to multi-cellular land plants requires 3006 new genes, but that transition from non-vascular to vascular plants is associated with a gain of only 516 genes [63]. They also reported that gene and genome duplication is pre-requisite for transition from simple leafless nonvascular sporophyte generation to dominant vascularized gametophyte generation and need almost three times as many new genes. The orthologous Physcomitrella and Selaginella shares around 84% to 89% of their genes in angiosperm plants, indicating their role as common ancestors [63]. These findings confirm that development from nonvascular to vascular life requires the stepwise addition of new genes directing their extra role in meristem development and hormonal signaling.

MAPK groups
In this study, we identified MAPK gene family members from different plant groups including monocots, dicots and lower eukaryotic organisms. Overall, the plants included six monocots, 26 dicots, five algae, one bryophyte, one pteridophyte, and one gymnosperm. The amplification and diversification of the large MAPK family in the monocot plant P. virgatum could be largely attributed to its tetraploid nature (2n = 4× = 36) [17]. It will be interesting to determine whether the paralogs in a single group have attained diverse functions in P. virgatum. Among the studied monocots, S. bicolour, S. italica and B. distachyon were found to have 16 member MAPK families. Similar numbers of MAPKs in Brachypodium have been reported by Chen et al. [18].
Group A MAPKs of monocot plants are very stable orthologs of both AtMPK3 and AtMPK6, whereas none of any monocot genomes harbor orthologs of AtMPK10, suggesting that this MAPK may have been lost before the split of monocots. Group B MAPKs of monocot plants posses several paralogs of MPK4, suggesting that the duplication of MPK4 paralogs might have occurred before divergence of monocot plants. Group C MAPKs in monocots contain paralogs of MPK7 and MPK14, but lack any MAPK1 and MAPK2 gene (Table 5). However, a recent study reported the presence of ZmMPK1 and ZmMPK2 in maize, which are putative orthologs of AtMPK1 and AtMPK2, respectively [15]. Liu et al. showed that ZmMPK1 and ZmMPK2 are phylogenetically closer to AtMPK1 and AtMPK2 than AtMPK7 and AtMPK14, respectively, and thus suggested that naming these MAPKs as ZmMPK1 and ZmMPK2 would be more appropriate. This group C MAPK was previously named as ZmMPK7 [74]. However, evaluation of the evolutionary relationship of monocot MAPKs indicated that it would be more appropriate to name these MAPKs as ZmMPK7 and ZmMPK14. Group D MAPKs of monocot plants showed the presence of 10/11 MAPK genes and are relatively constant as compared to the 15 MAPKs in P. virgatum (Table 1). These findings indicate that the amplification of monocot group D MAPKs occurred before diversification of this species.
Group A MAPKs, which are relatively constant in monocots with two or four members and in dicot plants, showed zero to seven MAPK members. M. guttatus, which is phylogenetically closer to Arabidopsis, do not possess any group A MAPKs (Table 5). Another plant, potato (S. tuberosum), is particularly conspicuous in that it lacks the group A MAPK from its genome. S. lycopersicum, another member of solanaceae family, has three group A MAPKs in its genome. The absence of complete group A MAPKs in M. guttatus and S. tuberosum is intriguing, and more work in these plant systems is needed to reveal whether other groups of MAPKs have acquired many intrinsic functions of group A MAPKs in these plant species. Another member of group A, an ortholog of AtMPK10, was only observed in B. rapa, T. halophila and C. rubella, which all belong to the mustard family. These findings suggest that MPK10 orthologs are conserved only in the brassicaceae family of dicots, while it was lost from other families (except Selaginella moelendorffii and Picea abies) during evolution ( Table 5).
The solanaceous plant S. tuberosum lacks group A MAPKs from their genome, suggesting that they have recently lost this group of MAPKs. The absence of group A MAPKs is particularly intriguing as it comprises orthologs of Arabidopsis MPK3 and MPK6. These two MAPKs show a high level of basal expression and even higher expression in response to biotic and abiotic stress, not only in Arabidopsis, but also in other plants. The roles of these two MAPKs in plant development have been well established in stomatal patterning [75], ovule development [76], seed formation and modulation of primary and lateral root development [77]. Definitive functions of group A MAPKs in growth and development and responses to biotic and abiotic stress have been shown in other plants as well [6,10,[78][79][80][81]. Furthermore, double mutants of mpk3 mpk6 show embryo lethality [10]. These studies in different plant systems suggest that MPK3 and MPK6 orthologs in different plant species are indispensable. It will be interesting to study these plants to address basic questions regarding MAPK signaling in response to different stress and developmental signals. Orthologs of AtMPK10 are confined to the dicot species B. rapa, T. halophila and C. rubella, which all belong to the mustard family. These findings suggest that MPK10 orthologs are only conserved in the brassicaceae family of dicots and have been lost from other families in the course of evolution ( Table 5). The presence of four BrMPK10 paralogs might be due to duplication after B. rapa speciation since other mustard family members have single orthologs of MPK10. Similar to MPK3 and MPK6 orthologs, group A MAPKs are either present together or absent. The paralogs of MPK3 and MPK6 have been reported in several plant species. A recent report identified MAPKs in canola (B. napus) shows absence of MPK10 in its genome [16]. It is interesting to note that B. napus (AACC) is an allotetraploid between B. rapa (AA) and B. oleracea (CC). Evaluation of the expression using the ATH1 GeneChip (at Arabidopsis thaliana Kinase Database-AthKD) under control and different abiotic stress conditions revealed significant expression of AtMPK10 only in three stages/tissues from 208 tissues/stress conditions. These findings suggest a limited role of AtMPK10 in Arabidopsis. However, it will be interesting to investigate the functional relevance of paralogs of MPK10 in B. rapa. For group B MAPKs, with the exception of A. thaliana, no orthologs of AtMPK11 was observed in any of the studied dicot plant species, even in the mustard family (Table 5). In Arabidopsis, AtMPK11 showed significant expression in several tissues and under various stress conditions, suggesting it plays a specific role and will not likely to be lost or evolve into a pseudogene. Similarly, MAPK5, another group B MAPK is only restricted to brassicaceae family members with the exception of being present in S. lycopersicum. Eight of ten group B MAPKs in G. max are paralogs of MAPK4. Such a high number of paralogs in soybean suggests extensive events of duplication might have occurred in its genome or part of the genome. These findings are concurrent with recent reports that showed G. max underwent at least two putative genome wide and/or segmental duplications approximately 13 and 59 million year ago [82][83][84][85][86].
The group C MAPKs members in dicot plants were ranges from one to six (Table 5). The single members of the MAPK group were found in three dicot species, E. grandis, M. guttatus and C. papaya, in the form of either MPK7 or MPK1. The four orthologs of Arabidopsis (AtMPK1, AtMPK2, AtMPK7, and AtMPK14) are not restricted to a particular family or species of dicot plants. The group D MAPKs are unique owing to their 'TDY' motif in the activation loop and long C terminal common docking domain (Rodriguez et al., 2010 [10]). In dicot plants, the number of group D MAPKs varies from 2 in A. coerulea to 14 MAPKs in B. rapa. Among the studied dicot species, 21 showed less than ten group D MAPKs, suggesting that amplification of this group of MAPKs is more prominent in monocots.
We previously discussed loss of group A MAPK MPK10 from monocots and all dicots except members of the brassicaceae family. The lower eukaryotic plant Selaginella and gymnosperm plant Picea abies were found to contain MPK10, a group A MAPK. These findings suggested that MPK10 were existed in older species and are lost during divergence and speciation, after which it was only able to be transferred to members of the brassicaceae family. To identify the evolutionary path of MAPKs, Doczi et al., analyzed MAPK signaling components in evolutionarily representative species of a plant lineage from a free-living amoebaflagellate protist, Naegleria (representative of an early diverging eukaryotic clade, Heterolobosea) to moss, algae and lycophytes [20]. Their study reported the presence of a single conventional MAPK in Naegleria with a 'TEY' motif, suggesting that the common ancestor of MAPKs were ERK-like, from which the distinct classes of MAPKs having TxY signature motifs were diverged [20]. Our study also showed multiple MAPKs (TEY, 2/3/4 and TDY, 1/2) in unicellular and multi-cellular algae ( Table 5), suggesting that diversification events of MAPKs started in very early periods of the evolution of photosynthetic eukaryotes.
As the complexity of organisms increased, the MAPK family showed expansion via gene duplication [20]. The P. patens, a model moss species with a genome of 487 Mb [87], contains eight MAPKs, six of which were TEY type and two were TDY type ( Table 5). The six TEY MAPKs were belonged to group B (four MAPKs) and group C (two MAPKs). Thus, on the evolutionary path of photosynthetic eukaryotes, lycophyetes, moss, multicellular and unicellular algae shows both TEY and TDY types of MAPKs. These findings suggest that the differences between these two types of MAPKs are ancient. Among mosses and algae, P. patens and O. lucimarinus lack 'group A' type MAPKs, while V. cartieri lacks 'group B' and 'group C' MAPKs, and C. reinhardtii and C. subellipsoidea do not possess 'group C' MAPKs. Interestingly, none of the studied species lacked 'group D' MAPKs. This observation suggests that the group D MAPKs or TDY MAPKs are indispensable in the lineage of green plants.
Overall, the MAPK gene family appears to be relatively constant in monocots (with the exception of P. virgatum), but not in dicots. Overall, the largest and smallest MAPK genes were observed in dicot plants.

Conclusion
Genome wide analysis of MAPK gene family revealed presence of new activation loop motifs. The adopted MAPK nomenclature system can be extended to other plant species to maintain uniformity in the MAPK nomenclature system. Presence of novel activation loop motifs are new variants and could offer new type of gene regulation in plants.

Identification of MAPK gene family members
Mitogen activated protein kinase (MAPK) gene families from the model plant Arabidopsis thaliana were downloaded from The Arabidopsis Information Resources (TAIR: http://www.arabidopsis.org/) database [88]. The MAPK gene families from rice were downloaded from the TIGR rice Genome Annotation Resources (http:// rice.plantbiology.msu.edu/) database [89]. The protein sequences of MAPKs from Arabidopsis thaliana and rice were used as search queries in the publicly available phytozome database (http://www.phytozome.net/) to identify MAPK genes in other plant species [90]. Overall, 40 species were included in this study and reported in Table 1. To identify MAPK gene families of unknown species, BLASTP searches was conducted using orthologous protein sequences Arabidopsis thaliana and Oryza sativa MAPK genes as the query search [91]. The genes identified through BLAST searches were used for further analysis. First, the top 100 genes were kept for systemic evaluation and indexing. The genes with serine/threonine protein kinase domains and the activation loop T-E-Y or T-D-Y motifs were considered as probable MAPK genes, which were subsequently confirmed by scanning in scan prosite and smart software for the presence of MAPK domain [92,93]. All datas were checked for redundancy and no any alternative splice variants were considered. Identified MAPK gene families from each species were again confirmed by running BLASTP searches against TAIR using the default parameters [92,93]. The genes were considered MAPK genes when BLASTP search matches with Arabidopsis MAPKs.

Multiple sequence alignment and construction of quaternary structure
Multiple sequence alignment of MAPKs were carried out using the multiple sequence alignment tool Multalin (http://multalin.toulouse.inra.fr/multalin/) to identify the conserved domains. The following default parameters were used to run the multiple alignment programs: Multalin: fasta, protein weight matrix: Blosum62-12-12, gap penalty at opening: default, gap penalty at extension: default, gap penalties at extremities: none, one iteration only: no, high consensus value: 90% (default), low consensus value: 50% (default), maximum line length: 180, and graduation step: 10. Molecular structures of AtMPK1 (group C), SiMPK20-1 (group D) and other MAPKs those contain novel activation loop motif were constructed by Phyre [94] software and resulted PDB file was subjected PYMOL to indicate activation loop motif of MAPKs.

Construction of phylogenetic tree
To construct the phylogenetic tree, all protein sequences were used to prepare a clustal file by running MAPKs protein sequences in clustal omega software using the following default parameters [95]: output format: clustal w/o numbers, dealign input sequences: no, MBED-like clustering guide tree: yes, MBED-like clustering iteration: yes, number of combined iterations: default (0), maximum guide tree iterations: default, maximum HMM (hidden Markov model) iterations: default, order: aligned. The generated clustal file of MAPKs was converted to MEGA file (.meg) format using the MEGA6 software and then employed to construct a phylogenetic tree [96]. The statistical parameters used to construct the phylogenetic tree were: analysis: phylogeny reconstruction; statistical method: maximum likelihood; test of phylogeny: bootstrap method; no. of bootstrap replications: 2000; substitution type-amino acids; model/method: Jones-Taylor-Thornton (JTT) model; rates among sites: uniform rates; gaps/missing data treatment: partial deletion; site coverage cutoff: 95%; ML heuristic method: nearest-neighbor-interchange (NII); initial tree for ML: make initial tree automatically (default-NJ/BioNJ); branch swap filter: very strong.

Statistical analysis
Tajima's relative rate test was carried out to understand the statistical significance between three different plant groups. The MEGA file used to construct phylogenetic tree was subjected to analyze Tajimas's relative rate test. Random MAPK sequences were selected to analyze Tajima's relative rate test in MEGA6 software. This random analysis was replicated for two times. Different statistical parameters used to analyze Tajima's relative rate test were; scope: for three chosen sequence, substitution type: amino acid gaps/ missing data treatment: complete deletion. On the other hand, Tajima's test of neutrality was carried out to understand the evolution of randomly evolved MAPKs from none randomly evolved MAPKs. Different statistical parameters used to analyze Tajima's test of neutrality were; scope: all