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  <cit> .
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  <cit> .
plant genomes are rich in genes that encodes protein kinases and constitute the kinase super-family  <cit> .
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  <cit> .
phosphorylated proteins carry out a wide array of cellular responses, including changes in gene expression, innate immunity, developmental programmes and stress and hormonal responses  <cit> .
mitogen activated protein kinases consist of three kinase-modules composed of mitogen activated protein kinase kinase kinase , mitogen activated protein kinase kinase  and mitogen activated protein kinase .
in the general model, extracellular signals activate mapkkks, which phosphorylate downstream mapkks.
the phosphorylated mapkks in turn phosphorylate mapks  <cit> .
protein phosphorylation events may occur throughout the protein kinase sequences, but usually occur on the activation loop  <cit> .
the activation loop, which is present at the c-terminal end, resides within sub-domain vii and viii of sub-domain eleven  <cit> .
the activation loop contains conserved serine, threonine and/or tyrosine amino acid residues that may be reversibly phosphorylated  <cit>  via cis auto-phosphorylation or trans phosphorylation by upstream kinases  <cit> .
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  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  <cit> , oryza sativa  <cit> , maize  <cit> , brassica napus  <cit> , apple  <cit> , and brachypodium  <cit> .
further, a study by janitza et al.
<cit>  and a review article by doczi et al.
<cit>  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  <dig> 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.
RESULTS
identification and nomenclature of mapks
we identified the mapk gene family from  <dig> 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  <dig> species collectively gave rise to  <dig> mapk sequences.
the tetraploid glycine max contained the most mapk genes in its genome , whereas the lower eukaryotic plant ostreocccus lucimarinus contained three .
in addition, brassica compestris , gossipium raimonddi , malus domestica , panicum virgatum , linum usitatissimum  and populus trichocarpa  contained higher number of mapks .
all the identified  <dig> mapks were provided with specific names according to the orthologous sequence similarity with arabidopsis thaliana or oryza sativa.table  <dig>
table representing genome size of different plant species and number of mapk genes present per genome
sl.
no
name of plant species
abbreviation of mapks
type of organism
ploidy level
genome size
total no.
of loci
total no.
of mapk genes
aquilegia coerulea
arabidopsis thaliana
brachipodium distachyon
brassica rapa
capsella rubella
carica papaya
chlamydomonas reinhardtii
citrus clememtina
citrus sinensis
coccomyxa subellipsoidea
cucumis sativus
eucalyptus grandis
fragaria vesca
glycine max
gossipium raimondi
linum usitatissimum
malus domestica
manihot esculenta
medicago truncatula
micromonas pusila
mimulus guttatus
oryza sativa
ostreococcus lucimarinus
panicum virgatum
phaseolus vulgaris
physcomitrella patens
picea abies
populus trichocarpa
prunus persica
ricinus communis
selaginella moellendorffii
setaria italica
solanum lycopersicum
solanum tuberosum
sorghum bicolor
thellungiella halophila
theobroma cacao
vitis venifera
volvox carteri
zea mays
from this table it is evident that, the number of genes in a specified gene family don’t directly proportional to its genome size.
genomics of mapks
among  <dig> mapks identified from  <dig> different plant species, fragaria vesca fvmpk <dig> contains the largest mapk gene, with  <dig> nucleotides long open reading frame , while panicum virgatum pvmpk1- <dig> has the smallest gene, with  <dig> nucleotides long orf .
transcript organization showed that mapks have different arrays of intron organization in their genes.
the numbers of mapks containing different arrays of introns were as follows: intronless , single intron , two introns , three introns , four introns , five introns , six introns , seven introns , eight introns , nine introns , ten introns  and eleven introns .
the terrestrial plant selaginella moellendorffii smmpk <dig> contained maximum of  <dig> introns in its gene.
some intronless mapks present in higher eukaryotic plants include pvmpk7- <dig>  pampk <dig>  pampk <dig>  pampk7- <dig>  and pampk <dig>  while lower eukaryotic algae contain olmpk <dig> and olmpk <dig> .
the molecular weights of mapk proteins were vary from  <dig>   to  <dig>   kda and the isoelectric points vary from  <dig>   to  <dig>   .
the isoelectric point  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  amino acid was maximum and tryptophan  amino acid was minimum .
the average abundance of the most important amino acids threonine, glutamic acid, and tyrosine  were  <dig> ,  <dig>  and  <dig> , respectively, whereas the average abundance of aspartic acid was  <dig> .
the abundance of the hydrophobic amino acids alanine , isoluecine , leucine , phenylalanine , proline  and valine  in mapks were relatively higher than that of other amino acids .
conserved motifs and domains
n-terminal conserved sequences
the mapks are characterized by the presence of a conserved t-e-y/t-d-y motif in the activation loop region.
despite having the activation loop t-e-y/t-d-y motif in mapks, in this study, we found that several mapks shared conserved n-terminal t-e-y, t-d-y, s-d-y and s-e-y motifs .
these n-terminal conserved motifs are only shared by group d mapks.
in total,  <dig> genes shared the n-terminal conserved motifs.
among them,  <dig> genes shared the s-d-y motif,  <dig> shared the s-e-y motif, six shared the t-d-y motif and the remaining <dig> genes shared the t-e-y motif .
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 .
several other group specific conserved motifs are also present in the n-terminal region of mapks.
they includes a-k-y, n-k-y , s-k-y, r-k-y , t-k-y  and s-q-y, n-r-y, s-r-y  .
these motifs are present immediately after the n-terminal t-e-y, t-d-y, s-d-y and s-e-y motifs.
the mapk sequences sharing different numbers of motifs are a-k-y , n-k-y , s-k-y , r-k-y , t-k-y , s-q-y , n-r-y , and s-r-y  .
in addition to the presence of conserved motifs, the n-terminal region of mapks also contained conserved amino acid consensus sequences including i-g-x-g-x-y-g-x-v, i-k-k-i-x3-f, d-a-x-r-x-l-r-e, f-x-d-i-y-x3-e-l-m, d-l-x2-v-i, d-x-l-x2-e-h, q-x-l-r-x-l-k-y-x-h, h-r-d-l-k-p-x-n, and l-x-l-x-n-c-x-l-k-i-x-d-f-g-l-a-r .figure  <dig>
multiple sequence alignment of plant mapks.
a. the figure shows presence of n-terminal tey, tdy, sey and sdy motifs .
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 atmpk <dig>  and simpk20- <dig> .
in atmpk <dig>  the arrow mark shows presence of tey motif in the activation loop region.
similarly, in the group d map kinase , the conserved n-terminal tey motif and activation loop tdy motifs are indicated by different arrows.
different conserved motifs present in n-terminal, activation loop region and c-terminal end of plant mapks
conserved signature motifs of mapksthe 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  <dig> and additional file  <dig>
table presenting different conserved consensus sequences present in plant mapks
common conserved signature sequences of mapks
n-terminal conserved consensus sequences
c-terminal conserved consensus sequences
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 t-e-y/ t-d-y motif in the activation loop region.
sequence alignment revealed that m. domestica mdmpk <dig> and p. trichocarpa ptmpk17-1contain an additional sequence of taykqyflwtklltfmkdy and tvcvflkpgftfqclidy between the conserved t-d-y motif .
in this study, we found eight novel activation loop motifs of mapks and reported here for first time.
the newly identified activation loop motifs are, t-q-y , m-e-y, t-e-c, t-v-y, , t-e-m , t-s-y, t-q-m, and t-r-m  .
in total, eight mapk genes share the m-e-y motif in the classic t-e-y and t-d-y region .
the mapk genes sharing the m-e-y motif are s. lycopersicum slmpk4- <dig>  s. tubersum stmpk4- <dig>  b. distachyon bdmpk4- <dig>  p. vulgaris fvmpk4- <dig>  s. italica simpk4- <dig>  z. mays zmmpk4- <dig>  s. bicolor sbmpk4- <dig> and o. sativa osmpk16- <dig>  these m-e-y motifs fall under group b mapks.
the new motif t-q-y  is shared by brmpk10- <dig>  t-e-c  by grmpk4- <dig>  t-v-y  by gmmpk4- <dig>  t-e-m  by pampk <dig>  pampk <dig> and pampk7- <dig>  while t-s-y is shared by olmpk <dig> , t-q-m  by pampk <dig> and t-r-m  by csubmpk <dig> .figure  <dig>
multiple sequence alignment of novel activation loop motifs of plant mapks.
beside presence of tey and tdy motifs in activation loop region, plant mapks show presence of other  <dig> different kinds of motifs  in the activation loop region as mentioned in figure .
these new motifs are aligned with conventional tey and tdy motifs of atmpk <dig> and atmpk <dig> in activation loop region.
this figure prepared to show the alignment of  <dig> newly identified motifs with tey and tdy activation loop motif of map kinase.
arabidopsis thaliana atmk <dig> and atmpk <dig> were taken as representative of tey and tdy motif respectively.
activation loop motifs are closely followed by presence of some group specific conserved motifs like dny, eey, sey, say, sky, sny, ggy etc.
these group specific motifs are indicated in blue color and present inside box.
molecular structure of plant mapks showing their activation loop motif.
the structures were modeled by homology based modeling procedure using the phyre <dig> server.
the activation loop motifs were labeled using pymol.
figures a, b, c, d, e, f, g, h, i, j represents t-e-y , t-d-y , m-e-y , t-e-m , t-s-y , t-e-c , t-v-y , t-q-y , t-q-m  and t-r-m  motif respectively.
c-terminal conserved sequences
the classical t-e-y/t-d-y motif at the activation loop region is followed closely by the presence of different c-terminal s-d-y/s-e-y/t-d-y/d-n-y/s-q-y/s-r-y/s-k-y/s-n-y motifs .
these s-d-y/s-e-y and t-d-y motifs are shared by group a and group b mapks.
additionally, the d-n-y, s-q-y, and t-k-y motifs are shared by group c mapks and s-k-y, t-k-y, s-r-y, and s-n-y motifs are shared by group d mapks .
the mapk sequences sharing different numbers of c-terminal motifs include s-d-y , s-e-y , t-d-y , d-n-y , s-q-y , s-k-y , s-r-y , and s-n-y .
there are also conserved t-r-w-y-r-a-p-e-l,i-d-x-w-s-v-g-c and q-x-l-l-x-f-d-p consensus sequences present in the immediately post activation loop region of mapks .
common docking domains
during mitogen activated protein kinase  signaling, the ability of map2ks to recognize their cognate mapks are facilitated by presence of short docking motif  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 .
the conserved docking domains of different mapks are k-m-l-t-f-d-p-k/r-q/k-r-i-t-v-e-d/e-a-l , k-m-l-v/i-f-d-p-x-k-r-i-i-v-d-e-a-l  and l-l-e-r/k-l-l-a-f-d-p-k-d-r-p-t-a-e-e-a-l  .table  <dig>
table showing predicted group specific common docking  sites of plant mapks
predicted common docking  sites of mapks
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.
in a. thaliana, mapk genes are classified into four different groups  based on their evolutionary relationship and presence of the t-d-y and t-e-y phosphorylation motif.
in this study, mapks are categorized into six different groups namely group a , b , c , d , e  and f  .
two new group of mapk  are generated during this analysis.
the new group e and f are mainly shared by mapks of lower eukaryotic and gymnosperm plants such as csubmpk <dig>  mpmpk <dig>  smmpk <dig>  creinmpk <dig>  vcmpk <dig>  csubmpk <dig>  pampk <dig>  pampk7- <dig>  pampk <dig>  pampk <dig>  creinmpk4- <dig>  vcmpk4- <dig>  olmpk <dig>  mpmpk <dig>  the phylogenetic analysis revealed that  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> mapks fall into group a, b, c, d, e, and f respectively .
the average overall phylogenetic mean distance of plant mapk is  <dig>  .
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.figure  <dig>
phylogenetic tree of mapk gene family in plants.
unrooted phylogenetic tree of mapks show presence of six different groups with well supported bootstrap values.
we named them as group a , b , c , d , e  and f .
the group e mapks are present at towards the end of the phylogenetic tree and group f present at the mid of the phylogenetic tree.
group e and f mapks are derived from lower plants of algae, pteridophytes and gymnosperm.
different mapk genes falls in different groups are; group a , group b , group c , group d , group e  and group f .
the erk <dig>  erk <dig>  atpin <dig>  and atcbl <dig>  were used as out group.
different statistical parameters used to constrcut the phylogenetic tree was: statistical method- maximum likelihood, test of phylogeny-boot strap method, no.
of bootsrap replicate- <dig>  model/method-jones-taylor-thornton  model, site coverage cutoff-95%, and branch swap filter-very strong.
phylogenetic tree was constructed using mega <dig> software.
table showing group specific distribution of different mapks in plants
sl.
no.
name of plant species
mitogen activated protein kinase
tey
tdy
other group
total
group a
group b
group c
group d
aquilegia coerulea
arabidopsis thaliana
brachipodium distachyon
brassica rapa
capsella rubella
carica papaya
chlamydomonas reinhardtii
citrus clememtina
citrus sinensis
coccomyxa subellipsoidea
cucumis sativus
eucalyptus grandis
fragaria vesca
glycine max
gossipium raimondi
linum usitatissimum
malus domestica
manihot esculenta
medicago truncatula
micromonas pusila
mimulus guttatus
oryza sativa
ostreococcus lucimarinus
panicum virgatum
phaseolus vulgaris
physcomitrella patens
picea abies
populus trichocarpa
prunus persica
ricinus communis
selaginella moellendorffii
setaria italica
solanum lycopersicum
solanum tuberosum
sorghum bicolor v  <dig>
thellungiella halophila
theobroma cacao
vitis venifera
volvox carteri
zea mays
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 .
more specifically, group c was used as out group.
when we took mgmpk4- <dig> , gmmpk16- <dig>  and atpin <dig> , resulted p-value was  <dig>  and x2-test result was  <dig>  .
when mgmpk4- <dig>  gmmpk16- <dig> and atcbl <dig> are taken as group a, b and c respectively, the p-value result was  <dig>  and x2-test result was  <dig> .
in both the cases statistical value was found to be significant .
the p-value less than  <dig>  is often used to reject the null hypothesis of equal rates between lineages .
the analysis involved  <dig> amino acid sequences.
all positions containing gaps and missing data were eliminated.
in tajima’s test statistics  for neutrality, the d value was found to be  <dig>   .
all the positions with less than 95% site coverage are eliminated during tajima’s test for neutrality.
there were a total of  <dig> positions in the final dataset.table  <dig>
tajima’s relative rate test
configuration
count
mgmpk4- <dig>  gmmpk16- <dig> atpin1
mgmpk4- <dig>  gmmpk16- <dig> atcbl1
x
tajima’s relative rate test was carried out by randomly comparing three phylogenetically distant sequences in each case by distributing them into three distinct group.
in column  <dig>  group a , b  and c ; column  <dig>  group a , b  and c ; column  <dig>  group a , b  and c ; column  <dig>  group a , b , and c .
test was replicated for four times.
in all the four cases, statistical result was found to be significant.
tajima’s test for neutrality
m
s
p
s
Θ
π
d
the analysis involved  <dig> 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  <dig> positions in the final dataset.
evolutionary analyses were conducted in mega <dig>  abbreviations: m = number of sequences, n = total number of sites, s = number of segregating sites, p
s = s/n, Θ = p
s/a <dig>  π = nucleotide diversity, and d is the tajima test statistic.
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.
the sizes of gene families reflect the number of duplicated genes, which are known as paralogs.
several plant mapks analyzed during this study were found to be duplicate genes resulting in several paralogous genes .
the plants with duplicated genomes give rise to more duplicated mapks relative to species with non duplicated genomes.
accordingly, species such as g. max, g. raimondii, and m. domestica contain more duplicated genes.
nevertheless, almost all species posses duplicated mapks in their genome.
the gene duplication result of plant mapks those contained novel activation loop motif are reported in table .
all the mapk resulted in z-score above four with 100% level of confidence.table  <dig>
gene duplication analysis of some selective plant mapks that contain novel activation loop motif
gene name
representative motif
z-score
level of confidence
analysis was carried out by pinda .
analysis shows all the mapks that contain novel activation loop motif resulted in z-score above four.
the z-score above four is considered highly significant to be duplicated.
mapk groups
mapks in monocotyledonous plants
among the  <dig> different plant species analyzed during this study, six were monocotyledonous plants  .
our study revealed that o. sativa contains  <dig> mapks, not  <dig> as reported earlier.
among monocot species, p. virgatum contains the highest number of mapks in its genome .
mapks in dicotyledonous plants
the mapk gene family of dicotyledonous plants has shown large variations among mapk gene family members .
among  <dig> different species investigated herein,  <dig> were dicot plants.
this group contained as few as  <dig> mapk gene in m. guttatus to as many as  <dig> in soybean .
investigation of mapks in g. max in another study using the hmm  approach also showed  <dig> mapks; however, four of them are indeed ‘mapk-likes’ genes, making the actual mapk number  <dig>
mapks in lower photosynthetic eukaryotes
the lower photosynthetic groups includes four algae, one bryophyte, one pteridophyte and one gymnosperm species .
multiple mapks were seen in several species of algae .
our study also showed multiple mapks  in unicellular and multi-cellular algae .
a genome survey of s. moellendorffii, a model lycophyte  and a primitive species revealed the presence of six mapks with single mapks in group a and b, whereas two mapks were present in group c and d. as a result, there are four mapks with the tey motif and only two with the tdy motif.
among the mosses and algae, p. patens and o. lucimarinus lacks ‘group a’ type mapks, while v. cartieri lacks ‘group b’ and ‘group c’ type mapks and c. reinhardtii and c. subellipsoidea do not possess ‘group c’ mapks.
interestingly, none of the studied lower photosynthetic eukaryotes or land plants  lacked ‘group d’ type mapks.
discussion
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  <dig> identified mapks across the plant lineage using the orthologous based nomenclature system proposed by hamel et al.
<cit> .
in the traditional naming system, names are assigned to gene that are identified and cloned first, regardless of their similarities to other gene.
for example, if someone cloned the thmpk gene from thelluginella halophila first, it named as thmpk <dig>  regardless of its orthologous similarity with other mpks.
accordingly, if this thmpk <dig> has orthologous similarity with atmpk <dig>  it should be named thmpk <dig>  but this does not happen.
however, orthology lends the legitimacy to transfer of functional similarities from its ancestors  <cit> .
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  <cit> , arabidopsis thaliana  <cit> , zea mays  <cit> , brachypodium distachyon  <cit> , canola   <cit>  and malus domestica  <cit> .
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 et al., revealed that the o. sativa genome contains  <dig> members of the mapk gene family  <cit> .
however, we recently identified  <dig> mapk gene family members from o. sativa.
additionally, zhang et al., reported  <dig> members of the mapk gene family from malus domestica  <cit> , but we found that m. domestica contains  <dig> members.
genomics of mapks
all species are confined by specific numbers of fundamental traits known as chromosome  <cit> .
the number of sets of chromosomes varies among genera and species, as well as within species.
some species are functionally haploid , or diploid  .
the genome size of a specific species is directly correlated with the ploidy level  of the organism  <cit> .
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  <cit> .
g. max encodes maximum of  <dig> mapks, whereas b. rapa encodes for  <dig> mapks.
similarly, m. domestica and g. raimondii encodes for  <dig> 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 .
during this study, we found that mapk genes harbored several introns .
additionally, different genes contained different numbers of introns ranging from zero  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  <cit> , and the presence of  <dig> mapk genes containing nine introns in their gene constitutes  <dig> % 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.
<cit>  these conserved motifs are target phosphorylation sites of upstream mapkk .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 .
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  <cit> .
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-x3-f, d-a-x-r-x-l-r-e, f-x-d-i-y-x3-e-l-m, d-l-x2-v-i, d-x-l-x2-e-h, q-x-l-r-x-l-k-y-x-h, h-r-d-l-k-p-x-n, and l-x-l-x-n-c-x-l-k-i-x-d-f-g-l-a-r .
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  reported that the protein kinase super-family contains the g-x-g-x-x-g conserved consensus sequence  <cit> , which is similar to the results of our study, in which i-g-x-g-x-y-g-x-v was conserved instead of g-x-g-x-x-g across all mapks .
in addition to the t-e-y/t-d-y activation loop motif of mapk, several new and novel activation loop motifs were identified those includes m-e-y , t-e-m , t-s-y , t-e-c , t-v-y , t-q-y , t-q-m  and t-r-m  .
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 s-d-y, s-e-y, t-d-y , d-n-y, s-q-y , s-k-y, and t-k-y  .
these motifs are may be putative phosphorylation sites of other kinases.
the presence of the n-terminal conserved activation loop motif immediately followed by the presence of another conserved motif greatly reflects presence of wide arrays of phosphorylation sites of mapks, which may be target phosphorylation sites of different other kinases.
the presence of n-terminal s-d-y/s-e-y/t-d-y motifs along with c-terminal s-d-y/s-e-y/t-d-y/s-q-y/d-n-y/s-r-y/s-k-y/t-k-y/a-k-y/s-n-y/g-r-y motifs reflects challenges to defining mapks; however, these mapks can now be better defined as group specific.
although it has been proposed that the t-e-y and t-d-y motifs at the activation loop are important for activation of mapks, the presence of n-terminal and c-terminal s-d-y/s-e-y/t-d-y as well 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 , hog , kss, fus, and smk <dig>   <cit> .
the cwi pathway is carried out by fungal mpk <dig> that contain t-e-y motif in its activation loop domain, hog pathway is carried out by hog <dig> that contain t-g-y motif, kss pathway is responsible for filamentous growth in fungi and kss <dig> 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 smk <dig> 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  <cit> ; 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  <cit> .
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  <cit> .
protein kinases often recognize their substrates and regulators through docking interaction sites that occurs outside the active site  <cit> , and these interactions can help us to understand the kinase networks.
the mapks contains group specific conserved docking domains, k-m-l-t-f-d-p-k/r-q/k-r-i-t-v-e-d/e-a-l , k-m-l-v/i-f-d-p-x-k-r-i-i-v-d-e-a-l , k-m-l-i-f-d-p-s/t-k-r-i-s-v-t-e-a-l  and l-l-e-r/k-l-l-a-f-d-p-k-d-r-p-t-a-e-e-a-l  .
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.
phylogeny of mapks
to understand the evolutionary expansion of mapks belonging to  <dig> different plant species, an unrooted tree was constructed from alignments of their full length protein sequences.
the analysis revealed that all mapks fell into six different groups with well supported bootstrap values.
we named them as group a , b , c , d , e  and f  .
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  of the phylogenetic tree .
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  <cit> .
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  <cit> .
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  <cit> .
expansion of the gene family may be involved in different developmental processes of plants  <cit> .
statistical analysis
in the random sampling of mapk sequences in tajima’s relative rate test, we found significant p-value and x2-test result in both the studied groups.
this implies that our study is statistically significant .
in tajima’s test for neutrality, the tajima’s d test result is  <dig>   .
in tajima’s d test, when d =  <dig>  the average heterozygosity became equal to number of segregating sites and can be interpreted as expected variation is similar to observed variation  <cit> .
the evolving population can be due to mutation-drift equilibrium and no evidence of selection.
when d <  <dig>  the average heterozygosity is lower than number of segregating sites  <cit> .
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 >  <dig>  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  <cit> .
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 + <dig> or less than − <dig> are considered as significant  <cit> .
in our result we found d value of  <dig>  which is greater than  <dig>  and + <dig> .
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  <dig> to  <dig>  mb that divided into n =  <dig> to n = ~  <dig> chromosomes  <cit> .
chromosomes evolved by fusion, fission, insertion, and duplication events, allowing evolution of chromosome size and number and hence the genes  <cit> .
vascular plants evolved approximately  <dig> million years ago and diverged into several lineages, among which lycophytes, ferns and gymnosperm and seed plant survived  <cit> .
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  <cit> .
the co-linearity resulting from the common ancestry of angiosperms provides a powerful method to determine the orthology  <cit> .
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 .
the most common mapk  that contain classical t-e-y motif in its activation loop motif is highly duplicated with z-score  <dig>  and 100% confidence level.
the atmpk <dig> that contained t-d-y motif in its activation loop motif resulted in z-score of  <dig>  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 z-score value four or more than four were considered as highly significant to be duplicated  <cit> .
the phylogenetic study showed that mapk genes of the grass family of monocotyledonous plants  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  <cit> .
it has also been reported that there are several duplicated mapk genes in grass family plants due to their ploidy level and genome duplication  <cit> .
vascular plants appeared approximately  <dig> million years ago, then diverged into several lineages  <cit> .
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  <cit> , a. thalaiana contains  <dig>   duplicated genes  <cit> .
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  <cit> .
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  <dig> new genes, but that transition from non-vascular to vascular plants is associated with a gain of only  <dig> genes  <cit> .
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  <cit> .
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,  <dig> 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   <cit> .
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  <dig> member mapk families.
similar numbers of mapks in brachypodium have been reported by chen et al.
<cit> .
group a mapks of monocot plants are very stable orthologs of both atmpk <dig> and atmpk <dig>  whereas none of any monocot genomes harbor orthologs of atmpk <dig>  suggesting that this mapk may have been lost before the split of monocots.
group b mapks of monocot plants posses several paralogs of mpk <dig>  suggesting that the duplication of mpk <dig> paralogs might have occurred before divergence of monocot plants.
group c mapks in monocots contain paralogs of mpk <dig> and mpk <dig>  but lack any mapk <dig> and mapk <dig> gene .
however, a recent study reported the presence of zmmpk <dig> and zmmpk <dig> in maize, which are putative orthologs of atmpk <dig> and atmpk <dig>  respectively  <cit> .
liu et al.
showed that zmmpk <dig> and zmmpk <dig> are phylogenetically closer to atmpk <dig> and atmpk <dig> than atmpk <dig> and atmpk <dig>  respectively, and thus suggested that naming these mapks as zmmpk <dig> and zmmpk <dig> would be more appropriate.
this group c mapk was previously named as zmmpk <dig>  <cit> .
however, evaluation of the evolutionary relationship of monocot mapks indicated that it would be more appropriate to name these mapks as zmmpk <dig> and zmmpk <dig>  group d mapks of monocot plants showed the presence of 10/ <dig> mapk genes and are relatively constant as compared to the  <dig> mapks in p. virgatum .
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 .
another plant, potato , 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 atmpk <dig>  was only observed in b. rapa, t. halophila and c. rubella, which all belong to the mustard family.
these findings suggest that mpk <dig> orthologs are conserved only in the brassicaceae family of dicots, while it was lost from other families  during evolution .
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 mpk <dig> and mpk <dig>  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  <cit> , ovule development  <cit> , seed formation and modulation of primary and lateral root development  <cit> .
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  <cit> .
furthermore, double mutants of mpk <dig> mpk <dig> show embryo lethality  <cit> .
these studies in different plant systems suggest that mpk <dig> and mpk <dig> 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 atmpk <dig> are confined to the dicot species b. rapa, t. halophila and c. rubella, which all belong to the mustard family.
these findings suggest that mpk <dig> orthologs are only conserved in the brassicaceae family of dicots and have been lost from other families in the course of evolution .
the presence of four brmpk <dig> paralogs might be due to duplication after b. rapa speciation since other mustard family members have single orthologs of mpk <dig>  similar to mpk <dig> and mpk <dig> orthologs, group a mapks are either present together or absent.
the paralogs of mpk <dig> and mpk <dig> have been reported in several plant species.
a recent report identified mapks in canola  shows absence of mpk <dig> in its genome  <cit> .
it is interesting to note that b. napus  is an allotetraploid between b. rapa  and b. oleracea .
evaluation of the expression using the ath <dig> genechip  under control and different abiotic stress conditions revealed significant expression of atmpk <dig> only in three stages/tissues from  <dig> tissues/stress conditions.
these findings suggest a limited role of atmpk <dig> in arabidopsis.
however, it will be interesting to investigate the functional relevance of paralogs of mpk <dig> in b. rapa.
for group b mapks, with the exception of a. thaliana, no orthologs of atmpk <dig> was observed in any of the studied dicot plant species, even in the mustard family .
in arabidopsis, atmpk <dig> 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, mapk <dig>  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 mapk <dig>  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  <dig> and  <dig> million year ago  <cit> .
the group c mapks members in dicot plants were ranges from one to six .
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 mpk <dig> or mpk <dig>  the four orthologs of arabidopsis  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 .
in dicot plants, the number of group d mapks varies from  <dig> in a. coerulea to  <dig> mapks in b. rapa.
among the studied dicot species,  <dig> 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 mpk <dig> 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 mpk <dig>  a group a mapk.
these findings suggested that mpk <dig> 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 amoeba-flagellate protist, naegleria  to moss, algae and lycophytes  <cit> .
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  <cit> .
our study also showed multiple mapks  in unicellular and multi-cellular algae , 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  <cit> .
the p. patens, a model moss species with a genome of 487 mb  <cit> , contains eight mapks, six of which were tey type and two were tdy type .
the six tey mapks were belonged to group b  and group c .
thus, on the evolutionary path of photosynthetic eukaryotes, lycophyetes, moss, multi-cellular 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 , but not in dicots.
overall, the largest and smallest mapk genes were observed in dicot plants.
CONCLUSIONS
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.

 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. 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. plant genomes are rich in genes that encodes protein kinases and constitute the kinase super-family. 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. phosphorylated proteins carry out a wide array of cellular responses, including changes in gene expression, innate immunity, developmental programmes and stress and hormonal responses. mitogen activated protein kinases consist of three kinase-modules composed of mitogen activated protein kinase kinase kinase, mitogen activated protein kinase kinase and mitogen activated protein kinase. in the general model, extracellular signals activate mapkkks, which phosphorylate downstream mapkks. the phosphorylated mapkks in turn phosphorylate mapks. protein phosphorylation events may occur throughout the protein kinase sequences, but usually occur on the activation loop. the activation loop, which is present at the c-terminal end, resides within sub-domain vii and viii of sub-domain eleven. the activation loop contains conserved serine, threonine and/or tyrosine amino acid residues that may be reversibly phosphorylated via cis auto-phosphorylation or trans phosphorylation by upstream kinases. 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 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, oryza sativa, maize, brassica napus, apple, and brachypodium. further, a study by janitza et al. and a review article by doczi et al. 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 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 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 species collectively gave rise to mapk sequences. the tetraploid glycine max contained the most mapk genes in its genome, whereas the lower eukaryotic plant ostreocccus lucimarinus contained three. in addition, brassica compestris, gossipium raimonddi, malus domestica, panicum virgatum, linum usitatissimum and populus trichocarpa contained higher number of mapks. all the identified mapks were provided with specific names according to the orthologous sequence similarity with arabidopsis thaliana or oryza sativa.table table representing genome size of different plant species and number of mapk genes present per genome sl. no name of plant species abbreviation of mapks type of organism ploidy level genome size total no. of loci total no. of mapk genes aquilegia coerulea arabidopsis thaliana brachipodium distachyon brassica rapa capsella rubella carica papaya chlamydomonas reinhardtii citrus clememtina citrus sinensis coccomyxa subellipsoidea cucumis sativus eucalyptus grandis fragaria vesca glycine max gossipium raimondi linum usitatissimum malus domestica manihot esculenta medicago truncatula micromonas pusila mimulus guttatus oryza sativa ostreococcus lucimarinus panicum virgatum phaseolus vulgaris physcomitrella patens picea abies populus trichocarpa prunus persica ricinus communis selaginella moellendorffii setaria italica solanum lycopersicum solanum tuberosum sorghum bicolor thellungiella halophila theobroma cacao vitis venifera volvox carteri zea mays from this table it is evident that, the number of genes in a specified gene family don’t directly proportional to its genome size. genomics of mapks among mapks identified from different plant species, fragaria vesca fvmpk contains the largest mapk gene, with nucleotides long open reading frame, while panicum virgatum pvmpk1- has the smallest gene, with nucleotides long orf. transcript organization showed that mapks have different arrays of intron organization in their genes. the numbers of mapks containing different arrays of introns were as follows: intronless, single intron, two introns, three introns, four introns, five introns, six introns, seven introns, eight introns, nine introns, ten introns and eleven introns. the terrestrial plant selaginella moellendorffii smmpk contained maximum of introns in its gene. some intronless mapks present in higher eukaryotic plants include pvmpk7- pampk pampk pampk7- and pampk while lower eukaryotic algae contain olmpk and olmpk. the molecular weights of mapk proteins were vary from to kda and the isoelectric points vary from to. the isoelectric point 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 amino acid was maximum and tryptophan amino acid was minimum. the average abundance of the most important amino acids threonine, glutamic acid, and tyrosine were, and, respectively, whereas the average abundance of aspartic acid was. the abundance of the hydrophobic amino acids alanine, isoluecine, leucine, phenylalanine, proline and valine in mapks were relatively higher than that of other amino acids. conserved motifs and domains n-terminal conserved sequences the mapks are characterized by the presence of a conserved t-e-y/t-d-y motif in the activation loop region. despite having the activation loop t-e-y/t-d-y motif in mapks, in this study, we found that several mapks shared conserved n-terminal t-e-y, t-d-y, s-d-y and s-e-y motifs. these n-terminal conserved motifs are only shared by group d mapks. in total, genes shared the n-terminal conserved motifs. among them, genes shared the s-d-y motif, shared the s-e-y motif, six shared the t-d-y motif and the remaining genes shared the t-e-y motif. 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. several other group specific conserved motifs are also present in the n-terminal region of mapks. they includes a-k-y, n-k-y, s-k-y, r-k-y, t-k-y and s-q-y, n-r-y, s-r-y. these motifs are present immediately after the n-terminal t-e-y, t-d-y, s-d-y and s-e-y motifs. the mapk sequences sharing different numbers of motifs are a-k-y, n-k-y, s-k-y, r-k-y, t-k-y, s-q-y, n-r-y, and s-r-y. in addition to the presence of conserved motifs, the n-terminal region of mapks also contained conserved amino acid consensus sequences including i-g-x-g-x-y-g-x-v, i-k-k-i-x3-f, d-a-x-r-x-l-r-e, f-x-d-i-y-x3-e-l-m, d-l-x2-v-i, d-x-l-x2-e-h, q-x-l-r-x-l-k-y-x-h, h-r-d-l-k-p-x-n, and l-x-l-x-n-c-x-l-k-i-x-d-f-g-l-a-r.figure multiple sequence alignment of plant mapks. a. the figure shows presence of n-terminal tey, tdy, sey and sdy motifs. 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 atmpk and simpk20-. in atmpk the arrow mark shows presence of tey motif in the activation loop region. similarly, in the group d map kinase, the conserved n-terminal tey motif and activation loop tdy motifs are indicated by different arrows. different conserved motifs present in n-terminal, activation loop region and c-terminal end of plant mapks conserved signature motifs of mapksthe 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  and additional file table presenting different conserved consensus sequences present in plant mapks common conserved signature sequences of mapks n-terminal conserved consensus sequences c-terminal conserved consensus sequences 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 t-e-y/ t-d-y motif in the activation loop region. sequence alignment revealed that m. domestica mdmpk and p. trichocarpa ptmpk17-1contain an additional sequence of taykqyflwtklltfmkdy and tvcvflkpgftfqclidy between the conserved t-d-y motif. in this study, we found eight novel activation loop motifs of mapks and reported here for first time. the newly identified activation loop motifs are, t-q-y, m-e-y, t-e-c, t-v-y,, t-e-m, t-s-y, t-q-m, and t-r-m. in total, eight mapk genes share the m-e-y motif in the classic t-e-y and t-d-y region. the mapk genes sharing the m-e-y motif are s. lycopersicum slmpk4- s. tubersum stmpk4- b. distachyon bdmpk4- p. vulgaris fvmpk4- s. italica simpk4- z. mays zmmpk4- s. bicolor sbmpk4- and o. sativa osmpk16- these m-e-y motifs fall under group b mapks. the new motif t-q-y is shared by brmpk10- t-e-c by grmpk4- t-v-y by gmmpk4- t-e-m by pampk pampk and pampk7- while t-s-y is shared by olmpk, t-q-m by pampk and t-r-m by csubmpk.figure multiple sequence alignment of novel activation loop motifs of plant mapks. beside presence of tey and tdy motifs in activation loop region, plant mapks show presence of other different kinds of motifs in the activation loop region as mentioned in figure. these new motifs are aligned with conventional tey and tdy motifs of atmpk and atmpk in activation loop region. this figure prepared to show the alignment of newly identified motifs with tey and tdy activation loop motif of map kinase. arabidopsis thaliana atmk and atmpk were taken as representative of tey and tdy motif respectively. activation loop motifs are closely followed by presence of some group specific conserved motifs like dny, eey, sey, say, sky, sny, ggy etc. these group specific motifs are indicated in blue color and present inside box. molecular structure of plant mapks showing their activation loop motif. the structures were modeled by homology based modeling procedure using the phyre server. the activation loop motifs were labeled using pymol. figures a, b, c, d, e, f, g, h, i, j represents t-e-y, t-d-y, m-e-y, t-e-m, t-s-y, t-e-c, t-v-y, t-q-y, t-q-m and t-r-m motif respectively. c-terminal conserved sequences the classical t-e-y/t-d-y motif at the activation loop region is followed closely by the presence of different c-terminal s-d-y/s-e-y/t-d-y/d-n-y/s-q-y/s-r-y/s-k-y/s-n-y motifs. these s-d-y/s-e-y and t-d-y motifs are shared by group a and group b mapks. additionally, the d-n-y, s-q-y, and t-k-y motifs are shared by group c mapks and s-k-y, t-k-y, s-r-y, and s-n-y motifs are shared by group d mapks. the mapk sequences sharing different numbers of c-terminal motifs include s-d-y, s-e-y, t-d-y, d-n-y, s-q-y, s-k-y, s-r-y, and s-n-y. there are also conserved t-r-w-y-r-a-p-e-l,i-d-x-w-s-v-g-c and q-x-l-l-x-f-d-p consensus sequences present in the immediately post activation loop region of mapks. common docking domains during mitogen activated protein kinase signaling, the ability of map2ks to recognize their cognate mapks are facilitated by presence of short docking motif 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. the conserved docking domains of different mapks are k-m-l-t-f-d-p-k/r-q/k-r-i-t-v-e-d/e-a-l, k-m-l-v/i-f-d-p-x-k-r-i-i-v-d-e-a-l and l-l-e-r/k-l-l-a-f-d-p-k-d-r-p-t-a-e-e-a-l.table table showing predicted group specific common docking sites of plant mapks predicted common docking sites of mapks 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. in a. thaliana, mapk genes are classified into four different groups based on their evolutionary relationship and presence of the t-d-y and t-e-y phosphorylation motif. in this study, mapks are categorized into six different groups namely group a, b, c, d, e and f. two new group of mapk are generated during this analysis. the new group e and f are mainly shared by mapks of lower eukaryotic and gymnosperm plants such as csubmpk mpmpk smmpk creinmpk vcmpk csubmpk pampk pampk7- pampk pampk creinmpk4- vcmpk4- olmpk mpmpk the phylogenetic analysis revealed that and mapks fall into group a, b, c, d, e, and f respectively. the average overall phylogenetic mean distance of plant mapk is. 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.figure phylogenetic tree of mapk gene family in plants. unrooted phylogenetic tree of mapks show presence of six different groups with well supported bootstrap values. we named them as group a, b, c, d, e and f. the group e mapks are present at towards the end of the phylogenetic tree and group f present at the mid of the phylogenetic tree. group e and f mapks are derived from lower plants of algae, pteridophytes and gymnosperm. different mapk genes falls in different groups are; group a, group b, group c, group d, group e and group f. the erk erk atpin and atcbl were used as out group. different statistical parameters used to constrcut the phylogenetic tree was: statistical method- maximum likelihood, test of phylogeny-boot strap method, no. of bootsrap replicate- model/method-jones-taylor-thornton model, site coverage cutoff-95%, and branch swap filter-very strong. phylogenetic tree was constructed using mega software. table showing group specific distribution of different mapks in plants sl. no. name of plant species mitogen activated protein kinase tey tdy other group total group a group b group c group d aquilegia coerulea arabidopsis thaliana brachipodium distachyon brassica rapa capsella rubella carica papaya chlamydomonas reinhardtii citrus clememtina citrus sinensis coccomyxa subellipsoidea cucumis sativus eucalyptus grandis fragaria vesca glycine max gossipium raimondi linum usitatissimum malus domestica manihot esculenta medicago truncatula micromonas pusila mimulus guttatus oryza sativa ostreococcus lucimarinus panicum virgatum phaseolus vulgaris physcomitrella patens picea abies populus trichocarpa prunus persica ricinus communis selaginella moellendorffii setaria italica solanum lycopersicum solanum tuberosum sorghum bicolor v thellungiella halophila theobroma cacao vitis venifera volvox carteri zea mays 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. more specifically, group c was used as out group. when we took mgmpk4-, gmmpk16- and atpin, resulted p-value was and x2-test result was. when mgmpk4- gmmpk16- and atcbl are taken as group a, b and c respectively, the p-value result was and x2-test result was. in both the cases statistical value was found to be significant. the p-value less than is often used to reject the null hypothesis of equal rates between lineages. the analysis involved amino acid sequences. all positions containing gaps and missing data were eliminated. in tajima’s test statistics for neutrality, the d value was found to be. all the positions with less than 95% site coverage are eliminated during tajima’s test for neutrality. there were a total of positions in the final dataset.table tajima’s relative rate test configuration count mgmpk4- gmmpk16- atpin1 mgmpk4- gmmpk16- atcbl1 x tajima’s relative rate test was carried out by randomly comparing three phylogenetically distant sequences in each case by distributing them into three distinct group. in column group a, b and c; column group a, b and c; column group a, b and c; column group a, b, and c. test was replicated for four times. in all the four cases, statistical result was found to be significant. tajima’s test for neutrality m s p s Θ π d the analysis involved 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 positions in the final dataset. evolutionary analyses were conducted in mega abbreviations: m = number of sequences, n = total number of sites, s = number of segregating sites, p s = s/n, Θ = p s/a π = nucleotide diversity, and d is the tajima test statistic. 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. the sizes of gene families reflect the number of duplicated genes, which are known as paralogs. several plant mapks analyzed during this study were found to be duplicate genes resulting in several paralogous genes. the plants with duplicated genomes give rise to more duplicated mapks relative to species with non duplicated genomes. accordingly, species such as g. max, g. raimondii, and m. domestica contain more duplicated genes. nevertheless, almost all species posses duplicated mapks in their genome. the gene duplication result of plant mapks those contained novel activation loop motif are reported in table. all the mapk resulted in z-score above four with 100% level of confidence.table gene duplication analysis of some selective plant mapks that contain novel activation loop motif gene name representative motif z-score level of confidence analysis was carried out by pinda. analysis shows all the mapks that contain novel activation loop motif resulted in z-score above four. the z-score above four is considered highly significant to be duplicated. mapk groups mapks in monocotyledonous plants among the different plant species analyzed during this study, six were monocotyledonous plants. our study revealed that o. sativa contains mapks, not as reported earlier. among monocot species, p. virgatum contains the highest number of mapks in its genome. mapks in dicotyledonous plants the mapk gene family of dicotyledonous plants has shown large variations among mapk gene family members. among different species investigated herein, were dicot plants. this group contained as few as mapk gene in m. guttatus to as many as in soybean. investigation of mapks in g. max in another study using the hmm approach also showed mapks; however, four of them are indeed ‘mapk-likes’ genes, making the actual mapk number mapks in lower photosynthetic eukaryotes the lower photosynthetic groups includes four algae, one bryophyte, one pteridophyte and one gymnosperm species. multiple mapks were seen in several species of algae. our study also showed multiple mapks in unicellular and multi-cellular algae. a genome survey of s. moellendorffii, a model lycophyte and a primitive species revealed the presence of six mapks with single mapks in group a and b, whereas two mapks were present in group c and d. as a result, there are four mapks with the tey motif and only two with the tdy motif. among the mosses and algae, p. patens and o. lucimarinus lacks ‘group a’ type mapks, while v. cartieri lacks ‘group b’ and ‘group c’ type mapks and c. reinhardtii and c. subellipsoidea do not possess ‘group c’ mapks. interestingly, none of the studied lower photosynthetic eukaryotes or land plants lacked ‘group d’ type mapks. discussion 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 identified mapks across the plant lineage using the orthologous based nomenclature system proposed by hamel et al.. in the traditional naming system, names are assigned to gene that are identified and cloned first, regardless of their similarities to other gene. for example, if someone cloned the thmpk gene from thelluginella halophila first, it named as thmpk regardless of its orthologous similarity with other mpks. accordingly, if this thmpk has orthologous similarity with atmpk it should be named thmpk but this does not happen. however, orthology lends the legitimacy to transfer of functional similarities from its ancestors. 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, arabidopsis thaliana, zea mays, brachypodium distachyon, canola and malus domestica. 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 et al., revealed that the o. sativa genome contains members of the mapk gene family. however, we recently identified mapk gene family members from o. sativa. additionally, zhang et al., reported members of the mapk gene family from malus domestica, but we found that m. domestica contains members. genomics of mapks all species are confined by specific numbers of fundamental traits known as chromosome. the number of sets of chromosomes varies among genera and species, as well as within species. some species are functionally haploid, or diploid. the genome size of a specific species is directly correlated with the ploidy level of the organism. 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. g. max encodes maximum of mapks, whereas b. rapa encodes for mapks. similarly, m. domestica and g. raimondii encodes for 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. during this study, we found that mapk genes harbored several introns. additionally, different genes contained different numbers of introns ranging from zero 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, and the presence of mapk genes containing nine introns in their gene constitutes % 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. these conserved motifs are target phosphorylation sites of upstream mapkk.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. 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. 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-x3-f, d-a-x-r-x-l-r-e, f-x-d-i-y-x3-e-l-m, d-l-x2-v-i, d-x-l-x2-e-h, q-x-l-r-x-l-k-y-x-h, h-r-d-l-k-p-x-n, and l-x-l-x-n-c-x-l-k-i-x-d-f-g-l-a-r. 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 reported that the protein kinase super-family contains the g-x-g-x-x-g conserved consensus sequence, which is similar to the results of our study, in which i-g-x-g-x-y-g-x-v was conserved instead of g-x-g-x-x-g across all mapks. in addition to the t-e-y/t-d-y activation loop motif of mapk, several new and novel activation loop motifs were identified those includes m-e-y, t-e-m, t-s-y, t-e-c, t-v-y, t-q-y, t-q-m and t-r-m. 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 s-d-y, s-e-y, t-d-y, d-n-y, s-q-y, s-k-y, and t-k-y. these motifs are may be putative phosphorylation sites of other kinases. the presence of the n-terminal conserved activation loop motif immediately followed by the presence of another conserved motif greatly reflects presence of wide arrays of phosphorylation sites of mapks, which may be target phosphorylation sites of different other kinases. the presence of n-terminal s-d-y/s-e-y/t-d-y motifs along with c-terminal s-d-y/s-e-y/t-d-y/s-q-y/d-n-y/s-r-y/s-k-y/t-k-y/a-k-y/s-n-y/g-r-y motifs reflects challenges to defining mapks; however, these mapks can now be better defined as group specific. although it has been proposed that the t-e-y and t-d-y motifs at the activation loop are important for activation of mapks, the presence of n-terminal and c-terminal s-d-y/s-e-y/t-d-y as well 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, hog, kss, fus, and smk. the cwi pathway is carried out by fungal mpk that contain t-e-y motif in its activation loop domain, hog pathway is carried out by hog that contain t-g-y motif, kss pathway is responsible for filamentous growth in fungi and kss 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 smk 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; 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. 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. protein kinases often recognize their substrates and regulators through docking interaction sites that occurs outside the active site, and these interactions can help us to understand the kinase networks. the mapks contains group specific conserved docking domains, k-m-l-t-f-d-p-k/r-q/k-r-i-t-v-e-d/e-a-l, k-m-l-v/i-f-d-p-x-k-r-i-i-v-d-e-a-l, k-m-l-i-f-d-p-s/t-k-r-i-s-v-t-e-a-l and l-l-e-r/k-l-l-a-f-d-p-k-d-r-p-t-a-e-e-a-l. 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. phylogeny of mapks to understand the evolutionary expansion of mapks belonging to different plant species, an unrooted tree was constructed from alignments of their full length protein sequences. the analysis revealed that all mapks fell into six different groups with well supported bootstrap values. we named them as group a, b, c, d, e and f. 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 of the phylogenetic tree. 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. 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. 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. expansion of the gene family may be involved in different developmental processes of plants. statistical analysis in the random sampling of mapk sequences in tajima’s relative rate test, we found significant p-value and x2-test result in both the studied groups. this implies that our study is statistically significant. in tajima’s test for neutrality, the tajima’s d test result is. in tajima’s d test, when d = the average heterozygosity became equal to number of segregating sites and can be interpreted as expected variation is similar to observed variation. the evolving population can be due to mutation-drift equilibrium and no evidence of selection. when d < the average heterozygosity is lower than number of segregating sites. 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 > 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. 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 + or less than − are considered as significant. in our result we found d value of which is greater than and +. 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 to mb that divided into n = to n = ~ chromosomes. chromosomes evolved by fusion, fission, insertion, and duplication events, allowing evolution of chromosome size and number and hence the genes. vascular plants evolved approximately million years ago and diverged into several lineages, among which lycophytes, ferns and gymnosperm and seed plant survived. 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. the co-linearity resulting from the common ancestry of angiosperms provides a powerful method to determine the orthology. 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. the most common mapk that contain classical t-e-y motif in its activation loop motif is highly duplicated with z-score and 100% confidence level. the atmpk that contained t-d-y motif in its activation loop motif resulted in z-score of 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 z-score value four or more than four were considered as highly significant to be duplicated. the phylogenetic study showed that mapk genes of the grass family of monocotyledonous plants 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. it has also been reported that there are several duplicated mapk genes in grass family plants due to their ploidy level and genome duplication. vascular plants appeared approximately million years ago, then diverged into several lineages. 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, a. thalaiana contains duplicated genes. 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. 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 new genes, but that transition from non-vascular to vascular plants is associated with a gain of only genes. 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. 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, 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. 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 member mapk families. similar numbers of mapks in brachypodium have been reported by chen et al.. group a mapks of monocot plants are very stable orthologs of both atmpk and atmpk whereas none of any monocot genomes harbor orthologs of atmpk suggesting that this mapk may have been lost before the split of monocots. group b mapks of monocot plants posses several paralogs of mpk suggesting that the duplication of mpk paralogs might have occurred before divergence of monocot plants. group c mapks in monocots contain paralogs of mpk and mpk but lack any mapk and mapk gene. however, a recent study reported the presence of zmmpk and zmmpk in maize, which are putative orthologs of atmpk and atmpk respectively. liu et al. showed that zmmpk and zmmpk are phylogenetically closer to atmpk and atmpk than atmpk and atmpk respectively, and thus suggested that naming these mapks as zmmpk and zmmpk would be more appropriate. this group c mapk was previously named as zmmpk. however, evaluation of the evolutionary relationship of monocot mapks indicated that it would be more appropriate to name these mapks as zmmpk and zmmpk group d mapks of monocot plants showed the presence of 10/ mapk genes and are relatively constant as compared to the mapks in p. virgatum. 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. another plant, potato, 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 atmpk was only observed in b. rapa, t. halophila and c. rubella, which all belong to the mustard family. these findings suggest that mpk orthologs are conserved only in the brassicaceae family of dicots, while it was lost from other families during evolution. 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 mpk and mpk 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, ovule development, seed formation and modulation of primary and lateral root development. 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. furthermore, double mutants of mpk mpk show embryo lethality. these studies in different plant systems suggest that mpk and mpk 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 atmpk are confined to the dicot species b. rapa, t. halophila and c. rubella, which all belong to the mustard family. these findings suggest that mpk orthologs are only conserved in the brassicaceae family of dicots and have been lost from other families in the course of evolution. the presence of four brmpk paralogs might be due to duplication after b. rapa speciation since other mustard family members have single orthologs of mpk similar to mpk and mpk orthologs, group a mapks are either present together or absent. the paralogs of mpk and mpk have been reported in several plant species. a recent report identified mapks in canola shows absence of mpk in its genome. it is interesting to note that b. napus is an allotetraploid between b. rapa and b. oleracea. evaluation of the expression using the ath genechip under control and different abiotic stress conditions revealed significant expression of atmpk only in three stages/tissues from tissues/stress conditions. these findings suggest a limited role of atmpk in arabidopsis. however, it will be interesting to investigate the functional relevance of paralogs of mpk in b. rapa. for group b mapks, with the exception of a. thaliana, no orthologs of atmpk was observed in any of the studied dicot plant species, even in the mustard family. in arabidopsis, atmpk 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, mapk 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 mapk 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 and million year ago. the group c mapks members in dicot plants were ranges from one to six. 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 mpk or mpk the four orthologs of arabidopsis 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. in dicot plants, the number of group d mapks varies from in a. coerulea to mapks in b. rapa. among the studied dicot species, 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 mpk 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 mpk a group a mapk. these findings suggested that mpk 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 amoeba-flagellate protist, naegleria to moss, algae and lycophytes. 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. our study also showed multiple mapks in unicellular and multi-cellular algae, 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. the p. patens, a model moss species with a genome of 487 mb, contains eight mapks, six of which were tey type and two were tdy type. the six tey mapks were belonged to group b and group c. thus, on the evolutionary path of photosynthetic eukaryotes, lycophyetes, moss, multi-cellular 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, but not in dicots. overall, the largest and smallest mapk genes were observed in dicot plants.  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. 
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. phylogenetic analysis of all predicted mapks were clustered into six different groups, and all predicted mapks were assigned with specific names based on their orthology based evolutionary relationships with arabidopsis or oryza mapks. mitogen activated protein kinase signaling is of critical importance in plants and other eukaryotic organisms. mitogen activated protein kinase activation loopconserved motifsorthologsparalogsphylogenyevolutionissue-copyright-statement© the author 2015 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, which are in turn are phosphorylated by map kinase kinase kinase. our study showed the presence of several new activation loop motifs and diverse conserved domains in 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. a total of mapks genes were identified from the genome wide analysis of species. 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.
 mitogen activated protein kinase 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, which are in turn are phosphorylated by map kinase kinase kinase. the mapks play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes.  a total of mapks genes were identified from the genome wide analysis of 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, 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mitogen activated protein kinase activation loopconserved motifsorthologsparalogsphylogenyevolutionissue-copyright-statement© the author 2015 