Genome-wide identification, phylogeny and expressional profiles of mitogen activated protein kinase kinase kinase (MAPKKK) gene family in bread wheat (Triticum aestivum L.)
- Meng Wang†1,
- Hong Yue†1,
- Kewei Feng1,
- Pingchuan Deng1,
- Weining Song1, 2Email author and
- Xiaojun Nie1Email author
© The Author(s). 2016
Received: 30 March 2016
Accepted: 3 August 2016
Published: 22 August 2016
Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the important components of MAPK cascades, which play the crucial role in plant growth and development as well as in response to diverse stresses. Although this family has been systematically studied in many plant species, little is known about MAPKKK genes in wheat (Triticum aestivum L.), especially those involved in the regulatory network of stress processes.
In this study, we identified 155 wheat MAPKKK genes through a genome-wide search method based on the latest available wheat genome information, of which 29 belonged to MEKK, 11 to ZIK and 115 to Raf subfamily, respectively. Then, chromosome localization, gene structure and conserved protein motifs and phylogenetic relationship as well as regulatory network of these TaMAPKKKs were systematically investigated and results supported the prediction. Furthermore, a total of 11 homologous groups between A, B and D sub-genome and 24 duplication pairs among them were detected, which contributed to the expansion of wheat MAPKKK gene family. Finally, the expression profiles of these MAPKKKs during development and under different abiotic stresses were investigated using the RNA-seq data. Additionally, 10 tissue-specific and 4 salt-responsive TaMAPKKK genes were selected to validate their expression level through qRT-PCR analysis.
This study for the first time reported the genome organization, evolutionary features and expression profiles of the wheat MAPKKK gene family, which laid the foundation for further functional analysis of wheat MAPKKK genes, and contributed to better understanding the roles and regulatory mechanism of MAPKKKs in wheat.
Mitogen-activated protein kinase (MAPK) cascades play the crucial role in plant growth and development as well as in response to stresses, which are highly conserved in the signal transduction pathway in eukaryote . The MAPK pathway included three main protein kinase members, namely MAPK kinase kinases (MAPKKK or MEKK), MAPK kinases (MKK or MEK) and MAPKs (MPK). They achieved the function through sequentially being phosphorylated. Upstream signals firstly activated the MAPKKKs, which in turn the MAPKKKs activated the MAPKKs and then specific MAPKs were activated by the MAPKKs. Eventually, the activated MAPKs phosphorylated transcription factors, enzymes or other signaling components to modulate the expression of downstream genes to complete signal amplification [2, 3]. It has been demonstrated that MAPK cascades played a vital role in cell division, growth and differentiation [4, 5], hormone response , plant immunity [7, 8], biotic and abiotic stress response and so on [9–11]. To date, extensive studies have been conduct to systematically investigate the MAPKKK gene family in many plant species and it is reported that there were 74 putative MAPKKK genes in maize (Zea mays), 75 in rice (O. sativa), 78 in cotton (G. raimondii) and 80 in Arabidopsis (A. thalianna), respectively [12–15].
Wheat is one of the most important crops worldwide, occupying 17 % of cultivated lands and serving as the staple food source for 30 % of the human population all over the world [16, 17]. Genetically, wheat is an allohexaploid species (2n = 6x = 42), which has a complex original and evolutionary history, derived from three diploid donor species through two naturally interspecific hybridization events. The initial hybridization event was occurred between A genome donor (T. urartu, AA; 2n = 14) and B geome donor (Aegilops speltoides, SS; 2n = 14) to produce the allotetraploid (AABB, T. turgidum L) about 0.2 MYa ago, and then the AABB donor crossed with the D genome donor (A. Tauschii Coss) to form the allohexaploid wheat (AABBDD) about 9000 years ago . As a result, wheat possesses a large and complex genome with three homologous genomes (A, B and D) and the size more than 17 Gb, which makes it a huge challenge to conduct genomic study in wheat. But, as the newly formed polyploidy, wheat is considered as an ideal model for chromosome interaction and polyploidization studies in plants [19, 20]. Recently, the draft genome sequencing of hexaploid wheat Chinese Spring (CS) was completed using the chromosome-based strategy, which laid the foundation to identify wheat gene family at the genome-level and also to discern the homologous copies in these three sub-genomes . The retention and dispersion of homologous gene will provide the indispensable information about chromosome interaction during polyploidization [21, 22].
At present, no systematical investigation of MAPKKK gene family has been performed in wheat. In light of the functional significance of this family, an in silico genome-wide search was conducted to identify wheat MAPKKK gene family in this study. Then, the chromosome localization, gene structure, conserved protein domain, phylogenetic relationship as well as expression profiles and regulatory network were systematically analyzed in the putative wheat MAPKKK genes to reveal the evolutionary and functional features of these genes. Our study will provide a basis for further functional analysis of the wheat MAPKKK genes, and will contribute to better understanding the molecular mechanism of MAPKKKs involving in regulating growth and development as well as stress processes in wheat.
Identification of MAPKKK gene family in wheat
The wheat MAPKKK gene family was identified following the method as described by Rao et al with some modifications . First, all the wheat protein sequences available were downloaded from the Ensemble database (http://plants.ensembl.org/index.html) to construct a local protein database. Then, this database were searched with 304 known MAPKKK gene sequences collected from A.thaliana (80), O. sativa (75), Z. mays (74) and B.distachyon (75) using the local BLASTP program with an e-value of 1e-5 and identity of 50 % as the threshold. Furthermore, all the MAPKKK sequences were aligned and the obtained alignments were used to construct a HMM profile using the hmmbuild tool embedded in HMMER3.0 (http://hmmer.org/download.html), and then the HMM profile were used to search the local protein database using the hmmsearch tool. HMMER and BLAST hits were compared and parsed by manual editing. Furthermore, a self-blast of these sequences was performed to remove the redundancy and the remaining sequences were considered as the putative TaMAPKKK proteins, which then were submitted to the NCBI Batch CD-search database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and PFAM databases (http://pfam.xfam.org/) to confirm the presence and integrity of the kinase domain. Finally, all the obtained sequences were verified the existence by BLASTN similarity search against the wheat ESTs deposited in NCBI database. The theoretical pI (isoelectric point) and Mw (molecular weight) of the putative TaMAPKKK were calculated using compute pI/Mw tool online (http://web.expasy.org/compute_pi/). Subcellular localization of each TaMAPKKK cascade kinases were predicted using the TargetP software of the CBS database .
Multiple sequence alignments and phylogenetic analysis
Multiple sequence alignments were generated using ClustalW tool . To investigate the evolutionary relationship among MAPKKK proteins, a neighbor-joining (NJ) tree was constructed by MEGA 6.0 software based on the full-length of MAPKKK protein sequences . Bootstrap test method was adopted and the replicate was set to 1000.
Gene structure construction, protein domain and motif analysis
The gene structure information were got from Ensemble plants database (http://plants.ensembl.org/index.html) and displayed by Gene Structure Display Server program (GSDS: http:/gsds.cbi.pku.edu.cn/). The protein domains and motifs in the MAPKKKs were predicted using InterProScan against protein databases (http://www.ebi.ac.uk/interpro/). The schematic representing the structure of all members of TaMAPKKKs was based on the InterProScan analysis.
Chromosomal locations and gene duplication
Genes were mapped on chromosomes by identifying their chromosomal position provided in the wheat genome database. Gene duplication events of MAPKKK genes in wheat were investigated based on the following three criteria: (a) the alignment covered >80 % of the longer gene; (b) the aligned region had an identity >80 %; and (c) only one duplication event was counted for the tightly linked genes [12, 26]. In order to visualize the duplicated regions in the T. aestivum genome, lines were drawn between matching genes using Circos-0.67 program (http://circos.ca/).
Identification of cis-regulatory elements
To investigate the cis-regulatory elements, the upstream regions (2 kbp) of all wheat MAPKKK genes were extracted, which were considered as the proximal promoter regions for the individual wheat MPKKK genes. Then, all the sequences were submitted to PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/Plantcare/html/) to identify the putative cis-acting regulatory elements.
Network interaction analysis
The interaction network which the TaMAPKKK genes involved were investigated based on the orthologous genes between Wheat and Arabidopsis using the AraNet V2 tool (http//www.inetbio.org/aranet/). Then, enrichment analysis was implemented by BiNGO, a cytoscape plugin, for gene ontology analysis and identifying processes and pathways of specific gene sets. Over-represented GO full categories were identified with a significance threshold of 0.01.
The MAPKKK gene expression analysis by RNA-seq data
To study the expression of TaMAPKKK genes in different organs and response to stress, transcriptome sequencing data obtained from WHEAT URGI (https://urgi.versailles.inra.fr/files/RNASeqWheat/) and NCBI Sequence Read Archive (SRA) database were used to investigate the differential expression of TaMAPKKKs. The accession numbers and sample information of the used data were listed in Additional file 1. TopHat and Cufflinks were used to analyze the genes’ expression based on the RNA-seq data . The FPKM value (fragments per kilobase of transcript per million fragments mapped) was calculated for each MAPKKK gene, the log10-transformed (FPKM + 1) values of the 155 TaMAPKKK genes were used for heat map generation. And fold change cutoff of two and p-value < 0.05, q-value < 0.05 were taken as statistically significant threshold [28, 29].
Plant materials, growth conditions, and treatments
The plants of wheat cultivar ‘CS’ were reared in growth chambers at 23 ± 1 °C with a photoperiod of 16 h light/8 h dark. The roots, stems, leaves, spikes (1 d before flowering), and grains (10d after pollination) were collected from flowering plants for tissue expression analysis. One-week-old seedlings which consisted with RNA-seq data were treated by 150 mM NaCl which represented salt treatment, and the seedlings grown under normal condition were used as control. The leaves of seedlings under salt and also control conditions were collected at 0, 6, 12, 24 and 48 h after treatment. All the plant samples from two biological replicates were frozen in liquid nitrogen immediately and stored at −80 °C for RNA isolation.
RNA isolation and qRT-PCR analysis
The total RNA was extracted using Plant RNA Kit reagent (Omega Bio-Tek, USA) according to the manufacturer’s instructions. The RNA integrity was checked by electrophoresis on 1.0 % agarosegels stained with ethidium bromide (EB). The first strand cDNAs were synthesized using a Vazyme Reverse Transcription System (Beijing, China) following the manufacturer’s protocol. Real-time PCR analyses were performed using the primer pairs listed in Additional file 2. Two biological and three technical replicates for each sample were obtained using the real-time PCR system (BIO-RAD CFX96, USA). The β-actin gene was used as internal reference for all the qRT–PCR analysis. Each treatment was repeated three times independently. The expression profile was calculated from the 2–△△CT value [ΔΔCT = (CTtarget/salt – CTactin/salt) – (CTtarget/control – CTactin/control)] .
Results and discussion
Genome-wide Identification of MAPKKK Family in Wheat
Comparison of the gene abundance in three subfamilies of MAPKKK genes in different plant species
Characteristics of the putative wheat MAPKKK genes
Ensemble Wheat Gene ID
Subfamily Gene ID
Amino acid length
Cytoplasmic Mitochondrial Nuclear
Cytoplasmic Mitochondrial Nuclear
Extracellular Cytoplasmic Nuclear
PlasmaMembrane Cytoplasmic Nuclear
To support the actual existence of these wheat MAPKKKs, we further performed a BLASTN search against the wheat expressed sequence tag (EST) and unigene database using the MAPKKKs as query. Results showed that most of the TaMAPKKKs’ existences were supported by EST hits except 6 MAPKKKs (TaMEKK4, TaMEKK13, TaMEKK25, TaRaf7, TaRaf53 and TaRaf98). We speculated these 6 not-support TaMAPKKKs might not express under any the used conditions or express with very low level that cannot be detected experimentally. Among the supported TaMAPKKK genes, TaRaf62 has the largest hits of ESTs, with the number of 119, followed by TaMEKK5 and TaRaf87 with the number of 95 and 55 ESTs, respectively.
Chromosome localization analysis found that the 155 TaMAPKKK genes were unevenly distributed on all the 21 wheat chromosomes, of which chromosome 3A contained the most MAPKKK genes with the number of 15, followed by 2A with the number of 14, then 5B, 5D as well as 7D all with the number of 11, while the chromosome 7B had the least MAPKKK gene, with the number of only 1. Furthermore, the length of putative TaMAPKKK proteins ranged from 149 to 1335 amino acids, with the putative molecular weight (Mw) ranging from 16.5 to 146.1 kDa and theoretical isoelectric point (pI) ranging from 4.55 to 9.33, respectively. The subcellular localization analysis found that a total of 51 TaMAPKKKs localized in nuclear, 42 localized in cytoplasmic and 32 localized in plasma membrane, while the remaining were predicted to be located in chloroplast, mitochondrial and extra-cellular (Table 2).
Phylogenetic and conserved domains analysis of TaMAPKKKs
Furthermore, the protein domains of these wheat MAPKKK genes were identified by searching against InterProScan databases (Fig. 2c). Results found that each cluster of the MAPKKKs classified by phylogenetic analysis shared the similar protein structure and domain composition, demonstrating that the protein architecture is remarkably conserved within a specific subfamily of MAPKKKs. Protein kinases have been demonstrated to play the crucial role in mediating process of protein phosphorylation, which widely occurred in most cellular activities . In this study, we found all the TaMAPKKK proteins contained a kinase domain (IPR000719), and most of them had the serine/threonine protein kinase active site (IPR008271) in the central part of the catalytic domain. These features were also found in the MAPKKK proteins of rice and cucumber [13, 33], suggesting the conserved function of MAPKKK genes in plants. Moreover, the ATP-binding site, which is located on the catalytic domain, is the most conserved sequences in the kinase family . We found that most of TaMAPKKKs also contained an ATP-binding site (IPR017441), suggesting that these wheat MAPK cascade kinases use ATP as the ligand in signal transduction pathway. In addition, the TaMAPKKKs also had some other conserved domains, such as concanavalin A-like lectin/glucanase domain (IPR013320), armadillo-like helical (IPR011989), and EF-hand domain (IPR011992). Interestingly, these TaMAPKKKs containing the same protein domains were generally clustered into the same clade in phylogenetic analysis, and showed similar expression patterns in response to multiple stresses, which was consistent with the result of BdMAPKKK genes as reported previously . For example, most TaMAPKKK genes containing concanavalin A-like lectin/glucanase domain were up-regulated by drought stress, while those genes containing armadillo-like helical domain showed to be down-regulated under salt stress. These results indicated that the various protein domains could regulate the TaMAPKKK gene to exhibit specific biological functions. The conserved domains identification and analysis may facilitate the identification of functional units in these kinase genes and accelerate to understand their crucial roles in plant growth and development as well as stresses response [34, 35].
Analyses of gene structures and promoter regions of TaMAPKKKs
Gene structure analysis can provide important information about the gene function, organization and evolution . Thus, the exon/intron structures of TaMAPKKK genes were further analyzed using the available wheat genome annotation information and then were displayed by the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) (Fig. 2b). We found the exon/intron structures in the TaMAPKKK genes were relatively conserved within the subfamily but some divergent between different subfamily. The Raf and MEKK subfamily have more sophisticated structure than ZIK subfamily due to the various number of intron. In detail, all the ZIK genes had introns, with the number ranging from 1 to 7. In the MEKK subfamily, 3 gene had no intron, and others had 1 to 22 introns, which was the most highly variable in the number of introns in TaMAPKKKs. In the Raf subfamily, 7 out 115 genes had no intron, and other Raf genes had the intron number ranging from 1 to 14. Interestingly, most gene pairs clustered together by phylogenetic analysis shared the similar exon/intron structure and intron phases in these TaMAPKKK genes, suggesting the evolutionary event may impact not only on the gene function but also on gene structure. It has been revealed that intron gain or loss is the results of selection pressures during evolution in plants, and the genes tend to evolve into diverse exon-intron structures and perform differential functions [37, 38]. Accordingly, the wheat MAPKKK genes were found to have the similar exon-intron structure within same subfamily, while the numbers of introns were varied, even within subfamily, which indicated that gene differentiation have occurred in the wheat MAPKKK to accomplish different biological functions under the selection pressure during the wheat genome formation and evolution.
Promoter is the region of the transcription factors (TF) binding site to initiate transcription, which plays a key role in regulating gene spatial and temporal expressions . To further detect the possible biological function and transcription regulation of these TaMAPKKKs, the 2 kb-upstream region of the transcriptional start site of all these genes were extracted and then used to screen for cis-regulatory elements. Results showed that a large number of stress-related and hormone-related cis-elements were found in promoter regions of the wheat MAPKKK genes (Additional file 3), which were similar with the result in Brachypodium, tomato and cucumber [32, 33, 36]. In addition, the abiotic stress-related (a total of 9 drought-stress, 1 salt-stress, 1 heat-stress, 1 cold-stress, 2 wound-stress and 2 disease resistance-related) and hormones signaling transduction-related (6 gibberellins, 4 abscisic acid and 3 ethylene-related) cis-regulatory elements were also found, suggesting that the wheat MAPKKKs may involve in regulating varieties of stress responses and hormone signaling transduction processes.
Genomic distribution and gene duplication of TaMAPKKK gene family
Gene duplication is frequently observed in plant genomes, arising from polyploidization or through tandem and segmental duplication associated with replication . In our study, a total of 11 homologous gene groups with a copy on each of A, B and D homologous chromosome were found in wheat MAPKKK gene family, and 24 gene pairs with a copy on only 2 of the 3 homologous chromosomes were also identified (Fig. 3 and Additional file 4), while the remaining 74 genes were not found homologs in wheat genome. Previous studies have demonstrated that the fractionation from ploidy caused the loss of some homologous sequences because of some combination of deletion . Our results indicated gene loss may also occur in wheat MAPKKK gene family, resulting in the loss of some homologous copies. The specific retention and dispersion of MAPKKKs in homologous chromosomes provide the invaluable information to better understand the wheat chromosome interaction and polyploidization. Furthermore, these homologous genes are clustered in group 2, 3 and 5 chromosomes, which was consistent with the above chromosome localization analysis, suggesting that group 2, 3 and 5 chromosomes suffered less sequence loss and interaction impact compared to other homologous chromosome groups.
Regulatory network between TaMAPKKK genes with other wheat genes
Tissue-specific expression patterns of TaMAPKKK genes
Expression patterns of TaMAPKKK genes under abiotic stresses
Validation of the expression of TaMAPKKKs by qRT-PCR analysis
This study for the first time identified and characterized the wheat MAPKKK gene family. Through a genome-wide search using the latest available wheat genome information, a total of 155 putative TaMAPKKKs were obtained, which classified into MEKK, ZIK and Raf 3 subfamilies based on the conserved motif signatures. The gene structure, conserved protein domain as well as phylogenetic relationship of these TaMAPKKKs were systematically analyzed and strongly supported the classification. The homologous genes between wheat A, B and D sub-genome and gene duplication were also investigated, which was found to be the main factors contributing to the expansion of wheat MAPKKK gene families. Furthermore, the expression profiles of wheat MAPKKKs during development and under abiotic stresses were investigated and the tissue-specific or stress-responsive TaMAPKKK genes were identified. Finally, 6 tissue-specific and 4 salt-responsive TaMAPKKK genes were selected to validate their expression level through qRT-PCR analysis, which provided the important candidates for further functional analysis of MAPKKK genes in wheat development and stress response. Our current study systematically investigated the genome organization, evolutionary features, regulatory network and expression profiles of the wheat MAPKKK gene family, which not only lay the foundation for investigating the function of these MAPKKKs, but also facilitate to reveal the regulatory and evolutionary mechanism of MAPK cascade involving in growth and development as well as in response to stresses in wheat.
This research was mainly funded by the National Natural Science Foundation of China (Grant NO: 31561143005 and 31401373), and partially supported by the 863 program (2012AA10A308) from the Chinese of Ministry of Science & Technology.
Availability of data and material
All of the datasets obtained from the public database and the data supporting the results of this article are included within the article and its Additional files. The phylogenetic data in our manuscript has been deposited into Treebase database with the accession No. S19638. The access URL is http://purl.org/phylo/treebase/phylows/study/TB2:S19638?x-access-code=e874b0f389ce8519b16789d764348e81&format=html.
NXJ and SWN designed the study and supervised the experiment. WM performed the bioinformatic analysis and prepared the manuscript. YH collected experimental materials. FKW and DPC conducted QPCR analysis. NXJ revised and improved the draft. All the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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