Genome-wide exploration of the molecular evolution and regulatory network of mitogen-activated protein kinase cascades upon multiple stresses in Brachypodium distachyon
© Jiang et al.; licensee BioMed Central. 2015
Received: 28 July 2014
Accepted: 9 March 2015
Published: 24 March 2015
Brachypodium distachyon is emerging as a widely recognized model plant that has very close relations with several economically important Poaceae species. MAPK cascade is known to be an evolutionarily conserved signaling module involved in multiple stresses. Although the gene sequences of MAPK and MAPKK family have been fully identified in B. distachyon, the information related to the upstream MAPKKK gene family especially the regulatory network among MAPKs, MAPKKs and MAPKKKs upon multiple stresses remains to be understood.
In this study, we have identified MAPKKKs which belong to the biggest gene family of MAPK cascade kinases. We have systematically investigated the evolution of whole MAPK cascade kinase gene family in terms of gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication analysis. Our results showed that most BdMAPK cascade kinases were located at the low-CpG-density region, and the clustered members in each group shared similar structures of the genes and proteins. Synteny analysis showed that 62 or 21 pairs of duplicated orthologs were present between B. distachyon and Oryza sativa, or between B. distachyon and Arabidopsis thaliana respectively. Gene expression data revealed that BdMAPK cascade kinases were rapidly regulated by stresses and phytohormones. Importantly, we have constructed a regulation network based on co-expression patterns of the expression profiles upon multiple stresses performed in this study.
BdMAPK cascade kinases were involved in the signaling pathways of multiple stresses in B. distachyon. The network of co-expression regulation showed the most of duplicated BdMAPK cascade kinase gene orthologs demonstrated their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolution analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provide valuable information for further investigation of the functions of BdMAPK cascade kinase genes.
Brachypodium distachyon is emerging as a widely recognized model plant of the grass subfamily Pooideae whose genome is completely sequenced . The plant has a very close relationship with several economically important Poaceae species such as Oryza sativa, Sorghum bicolor, Triticum aestivum and turf grasses. The study of the B. distachyon genome will help scientists better understand the mechanism of gene-controlled physiological processes in Poaceae, and subsequently improve the abiotic or biotic stress tolerance of crops and turf grasses through gene engineering.
Mitogen-activated protein kinases (MAPKs) cascades are evolutionarily conserved signaling modules, which are involved in controlling many cellular functions, including cell division, development and multiple stresses in all eukaryotes [2-4]. Activated MAPK cascade kinases can regulate the phosphorylation level of transcription factors and other components related to the MAPK pathway. For example, reactive oxygen species (ROS) can active MEKK1-MPK4 cascade, and the activated MAPK cascade further regulates the ROS-responsive gene expression and other MAPKs . Previous findings indicated that the MAPK cascade of MEKK1-MKK4/MKK5-MPK3/MPK6 was responsible for signal transmission when a plant recognized flagellin, and then triggered the disease resistant responses [6,7]. The MAPK cascade is not only involved in stresses responses, but also plays important roles in response to phytohormone. For instance, the MAPK cascade of MKK3–MPK6 has been proved as an important part of jasmonic acid (JA) signal transduction pathway in Arabidopsis thaliana [8,9]. Generally, a MAPK cascade contains three functionally conserved components: MAPKs, MAPK Kinases (MAPKKs/MKKs) and MAPKK Kinases (MAPKKKs/MEKKs). The external stimuli, once perceived by a membrane receptor, could translate them into cellular response signals, resulting in subsequent phosphorylation of MAPKKKs. The kinase-activation process of MAPKKKs initiates with the activation of MAPKKs by phosphorylating the serine and threonine residues in the S/TXXXXXS/T motif, then MAPKs at the last step of MAPK cascade through phosphorylation of both tyrosine and threonine residues in the TXY motif [4,10].
To date, numerous studies on plant MAPKs (also called MPKs) were conducted by many scientists . Similar to animal ERK kinases, plants MAPKs also have two phosphorylation motifs of TDY and TEY. A. thaliana MPKs, which have been reported to classify into four groups, are involved in developmental processes and the activation in response to biotic and abiotic stresses [12-14]. In A. thaliana, the groups of A and B including AtMPK3, AtMPK4 and AtMPK6 were well-characterized to respond to a diversity of environmental stimuli. For example, AtMPK4 was activated in a few minutes in response to the flg22 peptide of flagellin, and the activated AtMPK4 controlled the disease resistance gene expression and defense responses, showing a negative regulation in the biotic stress treatment [6,15]. AtMPK3 and AtMPK6 were involved in various environmental stress and hormone responses. Studies also revealed that MPK3 and MPK6 were activated when A. thaliana seedlings were treated with flg22. MPK3 and MPK6 were proved to be involved in other signaling pathways which are independent of MPK4 [6,7]. So far, ten MAPKKs in A. thaliana and eight MAPKKs in O. sativa have been found in response to multiple stresses. The upstream activation of AtMPK4 by AtMKK1 and AtMKK2 were involved in not only the ROS homeostasis and salicylic acid (SA) accumulation, but also abiotic stresses such as cold, salt and wounding [16-18]. OsMEK1, a homolog of AtMKK1, has been demonstrated to play an important role in response to the low-temperature stress at wide ranges in O. sativa . MAPKKKs (also named as MAP3Ks and MEKKs), known as the first step of MAPK cascade, constitute a diverse family of kinases which have been grouped into three large subfamilies based on the sequence of the kinase catalytic domain: the MEKK-like family, Raf-like family and ZIK-like family. Similar to mammalian MEKK1 and yeast STE11 and BCK1, plant MEKK-like subfamily members with a conserved catalytic domain are involved in stress responses by activating downstream MKKs . In A. thaliana, AtMEKK1, as a downstream kinase of the flagellin receptor FLS2, can trigger a complete plant MAPK cascade (MEKK1, MKK4/MKK5 and MPK3/MPK6), then function as a conservation innate immunity in response to both bacterial and fungal pathogens . The Raf-like family possesses more than half of MAPKKKs members, and the subfamily members share a specific polypeptide signature GTxx (W/Y) MAPE, which are similar to mammalian RAF1 [12,21,22]. The A. thaliana Raf-like members, Enhanced Disease Resistance 1 (EDR1) and Constitutive Triple Response 1 (CTR1), shared a homology with mammalian Raf-like MAPKKKs, were reported to participate in ethylene-mediated signaling and defense responses [23-26]. Sister clades such as ZIK-like kinases are presented in MAPKKKs phylogenetic analyses, however, these enzymes have not been shown to phosphorylate MAPKKs in plants [20,27].
So far, many members of MAPK cascades have been identified using functional genomic methods. Twenty MAPKs and 10 MAPKKs have been found in A. thaliana, 15 MAPKs and 8 MAPKKs are present in the O. sativa genome, while 16 MAPK genes and 12 MAPKK genes were identified from B. distachyon Bd21 genome . MAPKKK gene family has been systematically investigated in A. thaliana, O. sativa, Zea mays and Gossypium raimondii [21,22,29-31]. In this study, we have systematically identified all MAPK cascade kinase genes including a total of 75 MAPKKK genes from B. distachyon Bd21 genome. We further investigated the evolutionary relationship of B. distachyon MAPK cascade kinase genes (MAPKs, MAPKKs and MAPKKKs) in terms of phytogenetic analysis, chromosomal localization and gene duplication with their counterparts from monocot O. sativa and dicot A. thaliana. Subsequently, we used qRT-PCR to examine their tissue-specific transcription profiles and the profiles in response to several biotic or abiotic stresses. In addition, we analyzed the expression changes of BdMAPK cascade kinase genes under those treatments, and established the MAPK signaling network based on the co-expression patterns upon different stresses treatment. The duplicated ortholog pairs of BdMAPK cascade kinase genes revealed their convergent or divergent function in the process of evolution. Our study provided the genome-wide evolutionary analyses and expression profiles of MAPK cascade kinase genes in B. distachyon under multiple-stress conditions, which paved a way for further investigation into MAPK cascade kinase genes functions across different plant species.
Results and discussion
Distribution of MAPK cascade kinase genes in the plants
Identification and annotation of the MAPK cascade kinase gene family from the B. distachyon genome
The availability of a completed genome of B. distachyon offers the feasibility to identify all the MAPK cascade kinase gene family members in the plant. The identification and phylogenetic analyses of MAPK and MAPKK genes have been completed in B. distachyon previously, and therefore we used the same annotation of MAPK and MAPKK genes in this study as reported previously (Additional file 1) . In order to identify the MAPKKK genes, 155 queries of the MAPKKK sequences from A. thaliana and O. sativa were employed for BLASTP analyses against 32255 sequences of the protein database of B. distachyon available from MIPS (http://mips.helmholtz-muenchen.de/plant/brachypodium/), which retrieved 163 hits as target sequences . A self BLAST of these sequences followed by manual editing to remove the redundancy has finally identified 75 MAPKKK genes from the B. distachyon genome (Additional file 1). Furthermore, we analyzed the sequence homology between putative BdMAPKKK genes and the MAPKKK gene family in O. sativa using the Best Blast Mutual Hit (BBMH) method. Since there was no nomenclature rule of MAPKKKs to follow in A. thaliana and O. sativa, all 75 MAPKKK gene family members in B. distachyon genome were designated as BdMAPKKK1-BdMAPKKK75 based on the BBMH scores. The amino acid sequence analyses showed that all 75 BdMAPKKKs have a conserved protein kinase domain in the MAPK family. Protein subcellular localization was predicted by WoLF PSORT online analysis, and only the maximum probability was selected. The results revealed that most MAPK cascade kinases varied from the cytoplasm, chloroplast, and mitochondria to the nucleus except for 4 MAPKKKs and 4 MAPKs. For example, BdMAPKKK40 and BdMAPKKK75 were present in the cytoskeleton, whereas BdMAPKKK19 was localized in peroxisomes (Additional file 1).
Gene and protein structural organization of BdMAPKs, BdMAPKKs and BdMAPKKKs
Next, we analyzed the gene structure, exon position and phases of intron in BdMAPK cascade kinase genes, and 15 kinds of gene structures were identified (Figure 2B). Generally, the MAPK, MAPKK and MAPKKK genes could be divided into 3, 2 and 10 subgroups based on their exon/intron structures, respectively (Figure 2B). As shown in Figure 2B, all the members of the MAPK family had introns, whereas the members of groups C and D in the MAPKK family had no intron. In the Raf-like gene family, the number of introns varied from 1 to 15, whereas the number of exons varied from 2 to 11 in the ZIK-like gene family. Half of the MEKK group genes had only one exon and no intron. The results showed that the BdMAPK cascade kinase genes were consistent with their homologous genes in A. thaliana, O. sativa and Zea mays by gene structure analyses . Additionally, most gene pairs, which were clustered together by phylogenetic analysis, shared a similar exon/intron structure and intron phases. The conserved exon/intron structure and intron phases in the BdMAPK cascade kinase genes revealed the close evolutionary relationship among all four species, and supported their classification.
Furthermore, we have predicted the protein domains of the BdMAPK cascade kinase family using InterProScan against protein databases. The structure of all members of the BdMAPK cascade kinases was shown as a scheme in Figure 2C. Generally, each cluster of the BdMAPK cascade kinases by phylogenetic analysis shared a similar protein structure. All members of the BdMAPKs, BdMAPKKs and BdMAPKKKs contained a protein kinase domain (IPR011009). Most protein kinases catalyzed the transfer of a phosphate group from nucleoside triphosphates (often ATP) to specific amino acid residues of a protein substrate, resulting in a conformational change affecting protein function. The ATP-binding site, which is located on the N-terminal extremity of the catalytic domain, belongs to the most conserved sequences in the protein kinase family. Thus, most BdMAPKs, BdMAPKKs and BdMAPKKKs contained an ATP-binding site (IPR017441), which suggested that these BdMAPK cascade kinases used ATP as a ligand in signal transduction pathways. In the central part of the catalytic domain, almost all BdMAPK cascade kinases, except the group D in the BdMAPKs, contain the active site conserved aspartic acid residue (IPR008271), which is important for the catalytic activity of the enzyme. The catalytic domain and their structures in the BdMAPK cascade kinases are similar among proteins within subfamilies, demonstrating that the protein architecture is remarkably conserved within a specific subfamily. Moreover, despite all of BdMAPK cascade kinases contained a protein kinase domain, many of them contained other domains, such as ACT domain, PAS domain, and Ankyrin repeat containing domain. Interestingly, these BdMAPK cascade kinases which contained the same domain were clustered together to a clade, and showed similar expression patterns in response to multiple stresses treatment. For instances, most Phox/Bemlp domain containing BdMAPKKK genes showed to be down-regulated after PEG or H2O2 treatment, while the ACT domain containing BdMAPKKK genes were up-regulated by Cd2+. These results suggested that the BdMAPK cascade kinases exhibited different biological functions in response to various physiologic reactions. Because the biological functions of many MAPK cascade kinase genes in B. distachyon remain to be elucidated, the above findings may facilitate the identification of functional units in BdMAPK cascade kinase genes and lead to the discovery of their roles in plant growth and development.
Genomic distribution and gene duplication of BdMAPKs, BdMAPKKs and BdMAPKKKs
Comparative analysis of the phylogenetic ortholog genes of MAPKs, MAPKKs and MAPKKKs in B. distachyon, A. thaliana and O. sativa
To examine the evolutionary relationships between different MAPK cascade kinases in B. distachyon, A. thaliana and O. sativa, phylogenetic trees were constructed from alignments of the full MAPK cascade kinase amino acid sequences using the Neighbor-Joining (NJ) method by MEGA5.0 (Additional file 2). The gene model and amino acid sequences of MAPK cascade kinases in B. distachyon, A. thaliana and O. sativa were shown in Additional files 3 and 4. The phylogenetic analysis indicated that each of the BdMAPKs and BdMAPKKs can be divided into four subgroups in A. thaliana and O. sativa, which were consistent with the previous report , whereas MAPKKKs can be subdivided into three major subtypes, Raf-like family, MEKK-like family and ZIK-like family (Additional file 2). Moreover, Raf-like family contains more than half of MAPKKKs members in all three candidate plants. In general, all MAPK cascade kinases and their subgroups were present in monocots and dicots (Additional file 2), indicating that the occurrence of most components of the MAPK cascades in plants predates the monocot-dicot divergence and MAPK cascade kinase genes were conserved during evolution. Furthermore, MAPKKK phylogenetic tree showed similar clustering patterns in O. sativa and B. distachyon. In total, about 15 pairs of MAPKs, 8 pairs of MAPKKs and 60 pairs of MAPKKKs from O. sativa and B. distachyon were clustered as pairs, indicating that they might be the orthologous genes (Additional file 5). For instance, the amino acid sequence of BdMAPKKK29 and OsMAPKKK22 showed more than eighty percent of identities, indicating many consensuses in the MAPKKK protein sequences that may have existed before the species divergence between B. distachyon and O. sativa. The phylogenetic similarity found in O. sativa and B. distachyon suggested that they might have evolved conservatively. In contrast, B. distachyon has less orthologous genes than those in O. sativa compared to A. thaliana and a large number of MAPK cascade kinase genes were also clustered as pairs between B. distachyon and A. thaliana, suggesting that MAPK cascade kinase genes were large conserved gene families whose origin were very old (Additional file 5). The alignment of the conserved protein kinase domains showed that all Raf-like family in B. distachyon as well as O. sativa and A. thaliana shared Raf-like specific polypeptide signature GTxx (W/Y) MAPE, whereas ZIK-like subfamily contained a conserved polypeptide GTPEFMAPE (L/V/M)(Y/F) and MEKK-like members shared conserved polypeptide G (T/S) Px (W/F) MAPEV (Additional file 6).
Expression pattern of the BdMAPK, BdMAPKK and BdMAPKKK genes in different tissues
Differential expression profile of BdMAPK, BdMAPKK and BdMAPKKK gene upon multiple phytohormone treatments and abiotic or biotic stresses
In the following experiments, we demonstrated that MAPK cascades were not only involved in plant growth and development, but also played key roles in the control of plant response to multiple environmental stimuli including abiotic, biotic stresses and phytohormones. Firstly, the expression profiles of all gene members of the MAPK cascade kinases under different stress conditions were examined using qRT-PCR. A total of five abiotic stress types, i.e. heat, cold, NaCl, PEG and H2O2, which all can be activators of MAPK cascade pathway were tested in this study. Detailed expression profiles of the gene members of MAPK cascade kinases under different stress conditions were summarized in Additional file 8. Heat map representing the expression profiles of these gene members of MAPK cascade kinases under different stress conditions was shown in Figure 5D-F. Generally, our expression profile results were consistent with the Chen et al., who found that most of the MAPKs and MAPKKs were induced or constitutively expressed under stresses treatment . Similarly, our data revealed that 90% and 60% of MAPK cascade kinase genes were up-regulated under cold and heat stress conditions, respectively. Less MAPK cascade kinase genes were up-regulated under three other kinds of abiotic stress in this study, including NaCl, PEG and H2O2, which was also consist with Chen et al., who found that only 43.75% of MAPK family genes were up-regulated in the PEG treatment .
These results indicated that the temperature alteration is the most sensitive stress perceived by plants. Furthermore, a large number of MAPK cascade kinase genes were unchanged or slightly down-regulated at 3 h after treatment with heat, H2O2 and NaCl, wheaeas the genes were up-regulated at 6 hrs after treatments. Over a half of MAPK cascade kinase genes were up-regulated in both heat and cold treatments. A few MAPK cascade kinase genes were up-regulated under the conditions of all five kinds of abiotic stresses, such as BdMAPKKK51, −53, −58, −69 and so on. Most of the clustered gene pairs such as BdMAPKK3-1/3-2, MAPKKK2/65, MAPKKK6/12, and so on, showed the similar expression pattern after stress treatments, suggesting that these gene pairs might have similar physiological functions. On the other hand, several BdMAPKK gene pairs, which exhibited different expression patterns, may be involved in different signaling pathways. Furthermore, we also examined the expression profiles of all the gene members of MAPK cascade kinases to investigate whether the genes were involved in the response to heavy metal toxicity, including CdCl2, PbSO4 and ZnCl2 (Figure 5D-F). MAPK cascade kinase genes showed a very rapid increase in response to CdCl2 and PbSO4, while few MAPK cascade kinase genes were up-regulated by ZnCl2 treatment. In comparison with the abiotic stresses which have been discussed above, the expression level of BdMAPK and BdMAPKK genes (BdMKK1, BdMPK7-1, BdMPK16, BdMPK20-1, etc.) firstly increased within 6 hrs after heavy metal treatment and down-regulated at 12 hrs (Figure 5C and D). The same result was obtained in the expression pattern of most of BdMAPKKK genes which was in response to PbSO4 and ZnCl2 (Figure 5F). These results suggested that MAPK cascade kinases played a crucial role in response to heavy metal stress and the response was triggered within 6 hrs after stress treatment.
Recent studies of the MAPK cascades have also shown that the genes were responsive to JA, SA and ABA treatments. By treating plants with ABA, several components of MAPK signaling cascade genes showed a distinct inducible expression in many plant species, suggesting an important function of MAPK pathways in ABA signaling [36-39]. OmMKK1 was increased progressively in response to increasing lengths of exposure of MeJA, SA, ethephon and MV . To investigate the hormonal control mechanisms underlying MAPK cascade kinases, we treated Bd21 seedlings with three phytohormones, MeJA, SA and ABA, respectively and analyzed the changes of the transcription abundance of these MAPK cascade kinase genes using qRT-PCR. Our results demonstrated that 40% of MAPK cascade kinase genes were up-regulated by these three phytohormones after 3 hrs of treatment, respectively (Figure 5D-F). Only three of MAPK and MAPKK genes were up-regulated by all three phytohormones, whereas more than ten MAPKKKs were induced by MeJA, SA and ABA. It has reported that ABA as a phytohormone played an important role in integrating various abiotic stress signals and controlling downstream stress responses . Our data indicated that most BdMAPKKKs were showed a similar expression pattern under drough (PEG) and H2O2 stress conditions compared with ABA treatment, except for BdMAPKKK3, −4, −18, −27 and −73. These correlations of BdMAPKKKs expression levels between abiotic stress and phytohormone treatment suggested that the stress induced MAPK cascade signal transduction might be linked to the stress induced phytohormone alteration.
It has been reported that pathogen-associated molecular patterns (PAMPs)-triggered immunity requires a signal transduction from receptors to downstream components via the MAPK cascade, suggesting that plant MAPK cascades play a key role in the induction of defense mechanisms [11,15,42]. For example, the A. thaliana fls2 mutant was more susceptible than the wild type (WT) to infection by the virulent pathogen DC3000, and the WT showed an enhanced resistance to DC3000 after flg22 treatment . To investigate the mechanisms of MAPK cascades in disease defense, we determined the expression profiles of MAPK cascade kinase genes in B. distachyon after phytopathogen treatments. A total of three phytopathogens, including Fusarium graminearum (F0968) and two strains of Magnaporthe grisea (Guy11, avirulent ACE1 genotype; PH14, virulent ACE1 genotype) were used to inoculate Bd21 seedling in this study. The expression profiles of the MAPK cascade kinase genes at 4 hpi (hour post-inoculation) and 12 hpi were shown in Figure 5D-F. Only a few members of BdMAPK and BdMAPKK genes were up-regulated by phytopathogen treatment, whereas approximately 40% of BdMAPKKK genes were phytopathogen-induced. Interestingly, a large number of BdMAPKKK genes were induced faster by PH14 than by Guy11. For example, BdMAPKKK39, −47, −49 and −63 were up-regulated at 4 hpi after infection by PH14, but at 12 hpi, they were down regulated by PH14 and up-regulated by Guy11. These results were consistent with the previous report about the expression pattern of BdWRKY genes, suggesting that BdMAPKKK genes might play an important role with BdWRKYs in plant defense . The relationship between MAPK cascade kinases and WRKY transcript factors in phytopathogen-induced plant disease defense in B. distachyon should be further investigated.
Regulatory network of MAPK cascade kinase genes
The identification and characterization of all MAPK cascade kinase genes in a grass model plant B distachyon would facilitate a better understanding of the evolutionary processes and functions of these gene families. First, this study has completed identification of MAPKKK genes, the biggest upstream kinase gene family of the MAPK cascade in B. distachyon. Subsequently, this study has done systematic molecular evolutionary analysis of all MAPK, MAPKK and MAPKKK genes as a whole in B. distachyon comparing to O. sativa and A. thaliana. Furthermore, most importantly, this study has established a MAPK signaling co-regulation network to investigate the multiple-stress-driven interactions between BdMAPKs, BdMAPKKs and BdMAPKKKs upon different stresses treatment. Base on the regulation network, we found that most of duplicated BdMAPK cascade kinase gene orthologs showed their convergent function, whereas few of them developed divergent function in the evolutionary process. The molecular evolutionary analysis of identified MAPK family genes and the constructed MAPK cascade regulation network under multiple stresses provided useful information for further investigation of the functions of BdMAPK cascade kinase genes across different plant species.
To identify B. distachyon genes encoding MAPKKK proteins with a kinase domain, we performed a BLASTP search among 32255 sequences of the protein database of B. distachyon from MIPS (http://mips.helmholtz-muenchen.de/plant/brachypodium/) using 155 query MAPKKK sequences from A. thaliana and O. sativa . A self BLAST of these sequences followed by manual editing to remove the redundancy finally resulted in the identification of 75 MAPKKK genes. To verify the reliability of our results, all putative non-redundant sequences were assessed with UniProt (http://www.uniprot.org/) and SMART (http://smart.embl-heidelberg.de/) analysis, respectively. A total of 75 BdMAPKKK genes were found in B. distachyon (Additional file 1). All of these 75 putative MAPKKK gene family members in B. distachyon genome were designed as BdMAPKKK1-BdMAPKKK75 base on the BBMH scores between putative BdMAPKKK genes with MAPKKK gene family in O. sativa. The annotation of 16 MAPK genes and 12 MAPKK genes from B. distachyon Bd21 genome in Additional file 1 were made according to the previous research .
Sequence and phylogenetic analysis
To analyze the sequence of the 75 typical identified BdMAPKKKs, 12 MAPKKs and 16 MAPKs, we performed multiple alignment analyses of the kinase domains, sequence by ClustalW (www.ebi.ac.uk/clustalw/). A neighbor-joining (NJ) tree was constructed using the MEGA version 5 software, based on the alignment of MAPK cascade nucleotide or amino acid sequences in O. sativa, A. thaliana and B. distachyon. To determine the statistical reliability, we conducted bootstrap analysis with the following parameters: p-distance and pairwise deletion. Bootstrap analysis was performed with 1000 replicates. The data of the phylogenetic tree were deposited in Treebase Web (Accession URL: http://www.psort.org/).
Gene structure analysis
The information of BdMAPK cascade kinase genes, including accession number, chromosomal location, ORF length and exon-intron structure, were retrieved from the B. distachyon genome Database (http://www.brachypodium.org/). As well as the gene struc-tures of the BdMAPK cascade kinases were generated with the GSDS version 2.0 (Gene Structure Display Server 2: http://gsds.cbi.pku.edu.cn/).
Protein analysis of BdMAPK cascade kinases
The conserved domains and motifs in the MAPK cascade kinases was predicted using InterProScan against protein databases (http://www.ebi.ac.uk/interpro/). The schematic representing the structure of all members of BdMAPK cascade kinases was based on the InterProScan analysis results. Subcellular localization predictions of each of the BdMAPK cascade kinases were carried out using WoLF PSORT server (http://purl.org/phylo/treebase/phylows/study/TB2:S17106?x-accesscode=37158a103e4acaea162c89867a5c7061&format=html). The theoretical pI (isoelectric point) and Mw (molecular weight) of BdMAPK cascade kinases were carried out using Compute pI/Mw tool online (http://web.expasy.org/compute_pi/). Subcellular localization predictions of each of the BdMAPK cascade kinases were carried out using WoLF PSORT server (http://wolfpsort.seq.cbrc.jp/).
MAPKKK gene synteny analysis
The gene duplications within the MAPK cascade kinase gene family in B. distachyon, O. sativa and A. thaliana genomes were based on the information from the Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/index/locus). In order to visualize duplicated regions in the B. distachyon, O. sativa and A. thaliana genome, lines were drawn between matching genes using Circos-0.64 program (http://circos.ca/) .
Cluster analysis of expression data
The 2-week old seedlings (Bd21) were used for harvesting leaf, stem and root samples. The protocol of abiotic stresses treatment for Bd21 was adopted according with the previous work with some modifies . For phytohormone analysis, 2-week-old seedlings were treated in MS liquid medium containing 100 μM MeJA, 100 μM ABA, 1 mM SA and 20 μM 6-BA for 3 h or 6 h, respectively. For abiotic stress treatment, 2-week-old seedlings were treated in MS liquid medium containing 20% PEG, 200 mM NaCl and 10 mM H2O2 for 3 h or 6 h, respectively. For heavy metal stress treatment, 2-week-old seedlings were treated in MS liquid medium containing 100 μM ZnCl2, 100 μM PbSO4 and 100 μM CdCl2 for 6 h or 12 h, respectively. Cold and heat treatments were achieved by placing 2-week-old seedlings in MS liquid medium at 4°C or 45°C for 3 h or 6 h, respectively. For phytopathogen treatment, 2-week-old seedlings were sprayed with Fusarium graminearum (F0968) and two strains of Magnaporthe grisea (Guy11, avirulent ACE1 genotype; PH14, virulent ACE1 genotype) for 4 h or 12 h. The samples of each treatment were collected three biological replications. The BdMAPK cascade kinase genes array constituted of 103 primer-sets representing all members of the B. distachyon MAPK cascade kinase gene family. The primer-sets were listed in Additional file 10. The expression of the 103 BdMAPK cascade kinase genes was assessed upon the qPCR result analysis. Each qPCR experiment was repeated three separate times. The expression profile was calculated from the –ΔΔCT value [−ΔΔCT = (CTcontrol.gene – CTcontrol.actin) – (CTtreat.gene – CTtreat.actin)], obtained by PermutMatrixEN version 1.9.3 software, and shown by a green-red gradient. The data were statistically analyzed using OriginPro 7.5 software. The up-regulated genes were defined as a fold-change greater than 2 with p-value < 0.05 and a fold change of 0.5 or less was used to define down-regulated genes when the p-value < 0.05. All qPCR data were submitted to NCBI GEO dataset. The accession number is GSE66497.
Regulatory network construction
The expression data of BdMAPK, BdMAPKK and BdMAPKKK were clustered together to form an integrated expression profile by Cluster 3.0 software and visualized by using TreeView software. The MAPK cascade kinase genes, whose correlation coefficients of expression profiles were greater than 0.5, were clustered together as a set of co-expression regulatory MAPK cascade kinase genes under different kinds of treatment conditions, including biotic, abiotic, heavy metal stress and hormone treatment, respectively (Additional file 9). The line drawing which represented the co-expression regulatory network was constructed according to the data of co-expression regulatory MAPK cascade kinase genes.
We want to thank the contributors of the B. distachyon Genome Database, which was a convenient tool used to search for BdMAPKKK genes. This work was supported by Grant for Starting Package to Research Group of Plant Abiotic Stress and Plant Genome Evolution in Shanghai Chenshan Plant Science Research Centre, Chinese Academy of Sciences and Shanghai Chenshan Botanic Garden from Shanghai Landscaping Administrative Bureau (NO. F0112423, F0122415, F122423, F132426) and the Fund for National Key Laboratory of Plant Molecular Genetics (Y109Z11161).
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