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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
Journal of Integrative Agriculture
August 2012
2012, 11(8): 1227-1235
RESEARCH ARTICLE
Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.) LIAN Wei-wei1, 2*, TANG Yi-miao1*, GAO Shi-qing1, ZHANG Zhao1, ZHAO Xin1 and ZHAO Chang-ping1 Beijing Engineering and Technique Research Center of Hybrid Wheat, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, P.R.China 2 College of Life Science,Capital Normal University, Beijing 100048, P.R.China 1
Abstract Mitogen activated protein kinases (MAPK) cascades based on protein phosphorylation play an important role in plant growth and development. In this study, we have identified 15 putative members of the wheat MAPK gene (TaMPK) family through an in silico search of wheat expressed sequence tags (EST) databases based on the presence of amino acid sequence of Arabidopsis and rice MAPKs. Phylogenetic analyses of MAPKs from wheat, rice and Arabidopsis genomes have classified them into seven subgroups (A, B, C, D, E, F, and G). Using the available EST information as a source of expression data, the MAPK family genes from Triticum aestivum were detected in diverse tissues. Further expression analysis of the MAPKs in NCBI EST database revealed that their transcripts were most abundant in callus (20%), followed by leaf (12%) and inflorescence (12%). Most MAPK family genes showed some tissue specificity. Key words: wheat, MAPK, EST, phylogenetic analysis, profile
INTRODUCTION Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses (Ichimura 2002; Nakagami et al. 2005; Mishra et al. 2006). A classical MAPK pathway consists of three protein kinases, MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. Upstream signals activate MAPKKKs (such as the Raf and Mos proteins), which then phosphorylate MAPKKs at a conserved S/T-X3-5S/T (X denotes any amino acid) motif. MAPKKs in turn activate a specific MAPK, through phosphorylation of the conserved threonine and tyrosine residues at a conserved T-X-Y (X denotes any amino acid) motif
Received 16 March, 2011
located in the activation loop (T-loop) between kinase subdomains VII and VIII. The specifically activated MAPKs phosphorylate various downstream targets and regulate the growth, development, and stress responses of organisms (Schaeffer and Weber 1999; Fu et al. 2002; Larade and Storey 2006). To date, the best understanding of MAPKs was in yeast, at least 5 out of 6 MAPK genes were characterized (Gustin et al. 1998). However, experimental evidence has shown that MAPK cascades in plants play much wider regulatory roles than those in animals and yeast (Mizoguchi et al. 1997; Fu et al. 2002). In plants, several dozens of MAPKs have been identified and isolated from Avena sativa (Huttly and Phillips 1995), Petunia juss (Decroocq-Ferrant et al. 1995), Zea mays (Lalle et al. 2005), Arabidopsis thaliana
Accepted 28 June, 2011
LIAN Wei-wei, Mobile: 15210842024, E-mail: [email protected]; Correspondence ZHAO Chang-ping, Tel: +86-10-51503712, E-mail: [email protected] * These authors contributed equally to this work. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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(Mizoguchi et al. 1993), Hordeum vulgare (Knetsch et al. 1996), Nicotiana tabacum (Wilson et al. 1995), Oryza sativa (Fu et al. 2002), Chorispora bungeana (Zhang et al. 2006), respectively. During the past decade, incredible progress has been made towards the functional understanding of all genes in the model dicot Arabidopsis. In Arabidopsis genome, 20 MAPKs, 10 MAPKKs and 80 MAPKKKs were identified (Ichimura 2002). MAPK genes in rice (Oryza sativa) also were researched, and 17 members have been identified and analyzed (Reyna and Yang 2006). Wheat is one of the most important crops in the world. However, little is known about the MAPK genes and their regulatory roles in wheat. Up to date, only two TaMAPKs (WCK-1, FLRS) have been characterized in wheat (Takezawa 1999; Rudd et al. 2008). WCK-1 can be activated by fungal elicitors, which may involve Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts (Takezawa 1999). The WCK-1 and FLRS are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola (Rudd et al. 2008). In this study, we conducted an in silico search of wheat EST databases to further identify members of the wheat MAPK gene family. A total of 15 genes were identified and among which 11 were novel. Meanwhile, a phylogenetic tree was constructed based on their homologous relationships. The MAPKs were grouped into 7 different subfamilies. Furthermore, a large group of Triticum aestivum ESTs was analyzed to gain insights into the MAPK family gene expression among different tissues.
RESULTS Collection of wheat MAPK genes Availability of wheat EST sequences has made it possible for the first time to identify the MAPK family members in this plant species (Gill et al. 2004). After carefully surveying the database, 15 putative genes were defined as wheat MAPKs by in silico analysis based on the presence of amino acid sequence of Arabidopsis and rice MAPKs (Table 1). Further, Fig. 1 showed that each of the wheat MAPK proteins carried a T[DE]Y motif between the subdomains VII and VIII that was necessary for activation of MAPK, which powerfully supported their reliability as the members of the MAPK family. Wheat MAPKs were named TaMAPKs in order to distinguish them from those having been cloned in wheat. In addition, TaMAPKs were numbered from 1 to 15 according to the numerical order they appeared on the phylogenetic tree.
Phylogenetic analysis of TaMAPKs Up to date, two TaMAPKs were identified (Takezawa 1999; Rudd et al. 2008). Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1 (Takezawa 1999). FLRS and WCK-1 were associated with the resistance responses (Rudd et al. 2008). In Arabidopsis, MAPKs have been catego-
Table 1 List of MAPK genes in wheat grain Name TaMAPK1 TaMAPK2 TaMAPK3 * TaMAPK4 * TaMAPK5 * TaMAPK6 * TaMAPK7 * TaMAPK8 * TaMAPK9 * TaMAPK10 * TaMAPK11 TaMAPK12 * TaMAPK13 * TaMAPK14 TaMAPK15 *
UniGene number
Published MAPK
Accession number
T-Loop
AA length
Group
Ta.55196 Ta.23916 Ta.50588 Ta.54338 Ta.7205 Ta.14147 Ta.55675 Ta.4456 Ta.25413 Ta.13373 Ta.3070 Ta.18660 Ta.68359 Ta.6053 Ta.68224
WCK-1 FLRS
AF079318 AY173962
MAPK2B
DQ322666
MAPK1a
AY881102
TEY TEY TEY TEY TEY TEY TDY TDY TDY TDY TDY TDY TDY TDY TDY
369 403 387 376 369 369 548 598 696 613 549 494 490 578 498
B B A A C C D D D D E F F G G
Subcellular localization Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Periplasmic Periplasmic Periplasmic Periplasmicr Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic
, novel genes that we predicted according to silico cloning.
*
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
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Fig. 1 Protein sequence alignment of wheat MAPKs. Alignment was performed using the ClustalX2 program. The hosphorylation activation motif TxY(TEY and TDY) and kinase subdomains were indicated by box. It indicated that these TaMAPKs belonged to the MAPK gene families.
rized into four groups (A, B, C, and D) (Ichimura 2002). Using plant MAPKs from multiple plant species, phylogenetic analysis (Ligterink and Hirt 2001) suggests that plant MAPKs can be grouped into five groups (Fig. 2). Pairwise comparison and phylogenetic analysis of 20 AtMPKs and 17 OsMPKs indicated that these MAPKs can be divided into six groups (A, B, C, D, E, and F) (Reyna and Yang 2006). In order to investigate the phylogenetic relationships, the entire MAPK protein sequences were aligned by ClustalW and analyzed using MEGA4.0. In this study, the phylogenetic tree containing 20 AtMPKs, 15 TaMAPKs and 17 OsMPKs was used for further analysis. Here, these MAPKs can be divided into seven groups (A, B, C, D, E, F, and G). MAPKs in groups A, B, C contain the TEY motif in their activation site, whereas those in the D, E, F, and G groups contain the TDY activation motif (Fig. 1). Interestingly, wheat and rice contain more MAPKs with the TDY phosphorylation site than with the TEY motif. There are 9 TaMAPKs and 11 OsMAPKs containing the TDY motif whereas 6 TaMAPKs and 6 OsMAPKs belong to the TDY groups. In contrast, the Arabidopsis genome contains more MAPKs with the TEY phosphorylation site than with the TDY motif. 12 AtMAPKs have the TEY motif but only 8 AtMAPKs contain the TDY motif. Group A contains two wheat MAPKs (TaMAPK3 and TaMAPK4), which shows some similarity with 5 AtMPKs (AtMPK4, AtMPK12, AtMPK11, AtMPK5, and AtMPK13) and 2 OsMPKs (OsMPK2 and
OsMPK6). These MAPKs have been less well studied but appear to be involved in environmental stress responses and cell division. For example, disruption of the AtMPK4 gene that share high similarity with OsMPK2 and OsMPK6 by transposon insertion created a constitutive systemic acquired-resistance phenotype (Petersen et al. 2000). In the wild-type background, biochemical analysis using a MPK4-specific antibody revealed that both biotic and abiotic stresses induced activation of MPK4 (Ichimura et al. 2000; Desikan et al. 2001). Group B contains TaMAPK1 and TaMAPK2, which shares high similarity with OsMAPK5 and OsMAPK1, respectively, is known to be associated with a variety of environmental and hormonal responses. TaMAPK1 (WCK-1) can be activated by fungal elicitors and participate the defense response (Takezawa 1999). The first wheat MKP (Triticum turgidum L. subsp. durum) (accession no. EU502843), shares a highly homology with the WCK-1, interacts in vivo with TaMAPK1 and TaMAPK2 and controls their subcellular localization (Zaidi et al. 2010). Otherwise, the TaMAPK1 (WCK1) and TaMAPK2 (FLRS) are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola (Rudd et al. 2008). Group C includes two wheat MAPKs named TaMAPK5 and TaMAPK6 that shows high similarity with OsMPK4 and OsMPK3. Information on the group C MAPKs is limited, OsMPK4 (also known as OsMAPK4 or OsMSRMK3) has been shown to be tran-
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Fig. 2 Phylogenetic relationship of MAPKs. Phylogenetic analysis contains 20 AtMPKs, 15 TaMAPKs and 17 OsMPKs, and using MEGA4.0 to construct a neighbor joining (NJ) phylogenetic tree. These MAPKs can be divided into seven groups (A, B, C, D, E, F, and G). MAPKs in groups A, B, and C contain the TEY motif in their activation site, whereas those in the D, E, F, and G groups contain the TDY activation motif.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
scriptionally regulated by various abiotic stresses (Fu et al. 2002; Agrawal et al. 2003). Microarray analysis detected circadian-rhythm-regulated expression of AtMPK7 (Schaffer et al. 2001). Groups D, E, and F include MAPKs that have the TDY motif in their activation site and contain nine wheat MAPKs reported herein. Group D contains four wheat M A P K s ( Ta M A P K 7 , Ta M A P K 8 , Ta M A P K 9 , TaMAPK10). Five OsMPKs (OsMPK7, OsMPK8, OsMPK9, OsMPK10, and OsMPK11) and three AtMPKs (AtMPK18, AtMPK19, and AtMPK20) also belong to this group. The OsMPK8 (same as OsWJUMK1) gene is induced by various heavy metals, but its induction by pathogen attack has not been demonstrated previously (Fu et al. 2002). Group E contains a wheat MAPK named TaMAPK11 that shows high similarity with OsMPK14. OsMPK15 and AtMPK16 also belong to this group. Group F contains two TaMAPKs (TaMAPK12 and TaMAPK13) that shares high similarity with OsMPK17 and OsMPK16, respectively. Group G contains two wheat MAPKs named TaMAPK14 and TaMAPK15 that show high similarity with OsMPK12 and OsMPK13, respectively. Three Arabidopsis MAPKs (AtMPK9, AtMPK15, AtMPK8) also belong to this group. AtMPK8 and AtMPK9 possess a serine-rich and a glutamic-acid-rich region in their N-terminal regions, respectively (Ichimura 2002). The OsMPK12 gene (same as OsBWMK1) was shown to be transcriptionally induced by the rice blast fungus and wounding (He et al. 1999).
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Gene expression patterns of MAPKs To generate the patterns of expression of these MAPK family genes across various tissues and to assess the overall similarities and differences between transcriptomes of different tissues or organs, we performed a coordinated gene expression analysis of the wheat ESTs from different kinds of organs. The MAPK family genes were detected in all the ten types of plant tissues studied namely cell culture, flower, leaf, root, stem, callus, crown, inflorescence, sheath, and seed. Among ten types of plant tissues, transcripts of these MAPK family genes were shown to be most abundant in callus (20%), followed by leaf (12%) and inflorescence (12%) (Fig. 4). Some MAPK family genes were detected in other tissues as well, including sheath, root, cell culture, crown, stem, flower, and seed. To further characterize the pattern of expression of the single gene in different tissues, expression profiles of 15 MAPK
Structural comparison of wheat MAPKs TaMAPK proteins have a highly conserved kinase domain containing all fifteen subdomains that are characteristic of MAPKs. Like MAPKs in Arabidopsis and rice, the wheat MAPKs in groups A, B, and C have the TEY motif in the activation site. And a short C-terminus containing a common docking (CD) domain that consists of the sequence [LHY]Dxx[DE]EpxC (x represents any amino acid) serving as a docking site for MAPKKs, phosphatases, and protein substrates (Bardwell et al. 2003). Wheat MAPKs with the TDY motif do not have a CD domain but have a relatively long C terminal region (Fig. 3).
Fig. 3 Structural comparison of the 15 wheat MAPKs. The organization of the functional domains and motifs, including the phosphorylation motif (TxY) and CD domain of each MPAK, is shown in cartoon format. Scanning of the protein sequences for the presence of known motifs and domains was performed at PlantsP (http://plantsp.sdsc.edu/) (Gribskov et al. 2001).
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genes of wheat were examined by analyzing EST counts. Most of the MAPK family genes were detected in some tissues but not in others. TaMAPK13 exists in the whole plant, and has no expression profile database, so it has not been listed in the Fig. 5.
DISCUSSION Abiotic and biotic stress conditions such as cold, drought and pathogens attack seriously affect wheat growth and yield. To improve wheat tolerance to these
Fig. 4 Distribution of wheat MAPK family genes in various tissues.
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abiotic and biotic stresses, researches have been focused on the physiological and molecular mechanisms of wheat responses to these stresses. Isolation and identification of members of the MAPK family from wheat is of great importance for better understanding the molecular genetic basis for the wheat genetic improvement and providing the functional genetic resources required for transgenic research. Here, a total of 15 TaMAPKs were identified, which is fewer than those currently reported in Arabidopsis (23 members) and rice (17 members) (Ichimura 2002) because the whole genome of wheat is not sequenced and EST databases is limited. While, it is possible that more TaMAPKs may be identified by more wheat sequence (EST and genome) information in the future, it is likely that we have identified most, if not all, of the MAPKs in wheat. MAPKs phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK-mediated signaling is required both for patterning and for cell proliferation in the developing Drosophila wing (Marenda et al. 2006), which suggested the importance of subcellular localization of MAPKs. Based on prediction subcellular
Fig. 5 Tissue specific expression of the MAPK family genes.
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
localization of the MAPK proteins using the CELLO online software, we found TaMAPK7, TaMAPK8, TaMAPK9, and TaMAPK10 were mainly located in p e r i p l a s m i c , w h i l e o t h e r Ta M A P K s w e r e i n cytoplasmic, the mechanism of which was not yet known. In this study, according to phylogenetic analyses of MAPKs from wheat, rice and Arabidopsis, seven subgroups (A, B, C, D, E, F, and G) were found (Fig. 3), The A, B and C groups of the MAPKs contain the TEY phosphorylation site, whereas the D, E, F, and G groups have the TDY motif. Interestingly, there are fewer TaMAPKs in the A, B, and C groups (6 members) than in the D, E, F, and G groups (9 members). It is possible that this ratio (6 TEY TaMAPKs vs. 9 TDY TaMAPKs) may change with the discovery of more new TaMAPKs from the wheat genome. MAPKs within the same group might function similarly. For example, group A MAPKs were most frequently involved in environmental stress responses and cell division, whereas group B appeared to be associated with a variety of environmental and hormonal responses. Some of MAPKs in group C participated in various abiotic stresses regulation. On the other hand, group D, E, F, and G MAPKs were larger in size than other group MAPKs and little was known about the function of their long Cterminal extension. MAPKs ensure their signalling specificity of action by interacting with their substrates through common docking domains (CD domain). These docking domains lead the kinases to the correct substrates and enhance their fidelity and efficiency of action (Sharrocks et al. 2000), which was also observed in wheat groups A, B, and C MAPKs (Tanoue et al. 2000). However, the CD domain alone does not determine the docking specificity. Tanoue et al. (2001) found that a docking groove in the steric structure of MAPKs, which comprised the CD domain and the ED site, serving as a common docking region for various MAPK-interacting molecules. Docking sites are specific and modular, which are important for phosphorylation. Therefore, the existence of this conserved CD domain (i.e., docking sites) will help us understand the mechanism of interactions between MAPKs and a variety of other proteins in wheat. ESTs are created by partially sequenced gene tran-
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scripts and have proved valuable in molecular biology. ESTs have played an important role in gene discovery and in analyzing the patterns of gene expression (Fei et al. 2004). Previous study demonstrated that some MAPK family genes exhibited tissue specificity. The expression of RsMPK2 (a MAPK family gene from R. soongorica) was detected in vegetative (root, stem, leaf and callus) and reproductive (flower) organs, but the patterns of gene expression is diversity (Liu et al. 2010). A wheat MAPK gene, TaMAPK13, was expressed in all tissues abundantly, whereas the TaMAPK15, only expressed in inflorescence at a low level. To detect the pattern of expression of MAPK family genes, we analyzed the expression of these genes in various tissues. The result revealed that MAPK genes of wheat were strictly controlled in different organs and at different growth stages. These results will be helpful in studying biological functions of MAPK family genes.
CONCLUSION The present study has, for the first time, provided a comprehensive list of MAPKs present in wheat. In silico search of EST databases using BLASTP resulted in identification of 15 MAPK genes from wheat and among which 11 were new. These findings provide new insights into the diversity and structure of MARK genes in wheat and the biological function and evolution of these genes.
MATERIALS AND METHODS Identification of wheat TaMAPKs In order to isolate MAPK family genes in wheat, the amino acid sequence of 23 Arabidopsis and 17 rice MAPK genes were used as a query to search the database of UniGene (ftp://ftp.ncbi.nih.gov/repository/UniGene/ Triticum_aestivum/Ta.seq.all.gz), using the tBLASTn program at an e-value of 1e-3 to avoid false positives, then assemble the searched MAPK genes using the CAP3 sequence assembly program (http://pbil.univlyon1fr./cap3. php) (Huang and Madan 1999). ORFs were performed with the ORF Finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/ gorf. html).
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Conserved domain detection and subcellular localization prediction Programs SMART, MOTIF SCAN, and PLANTSP were used to detect conserved domains. In brief, the MAP kinase signature, PK domain, ATP-binding domain and so forth were found in a given protein sequence, which can be regarded as a candidate of wheat MAPK family. Prediction on subcellular localization of wheat MAPKs was carried out using the CELLO ver. 2.5 server (http://cello.life. nctu.edu.tw/).
Sequence alignment and phylogenetic analysis A multiple alignment analysis was performed with Clustal W. Phylogenetic trees were constructed using the neighborjoining (NJ) method and diagrams of phylogenetic trees were drawn with the help of MEGA4, the parameters were set to “multiple alignment gap opening penalty was 10, gap extension penalty was 0.2, delay divergent cutoff was 30%”.
Analysis of expression profile The expression profile was suggested by analyzing the EST counts based on UniGene (http://www.ncbi.nlm.nih. gov/UniGene/UGOrg.cgi?TAXID=3711).
Acknowledgements This work was supported by the Genetically Modified Organisms Breeding Major Projects, China (2008ZX08002002, 2008ZX08002-003, 2008ZX08002-004), the Beijing Technical Nova Project, China (2007B056, 2008B035), the Excellence Scholar Fostered Program of Beijing Government, China (20081D0200500050), the Beijing Natural Science Foundation of China (5102016) and Young Foundation Project of Beijing Academy of Agriculture and Forestry Scientific Research, China.
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
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August 2012
2012, 11(8): 1227-1235
RESEARCH ARTICLE
Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.) LIAN Wei-wei1, 2*, TANG Yi-miao1*, GAO Shi-qing1, ZHANG Zhao1, ZHAO Xin1 and ZHAO Chang-ping1 Beijing Engineering and Technique Research Center of Hybrid Wheat, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, P.R.China 2 College of Life Science,Capital Normal University, Beijing 100048, P.R.China 1
Abstract Mitogen activated protein kinases (MAPK) cascades based on protein phosphorylation play an important role in plant growth and development. In this study, we have identified 15 putative members of the wheat MAPK gene (TaMPK) family through an in silico search of wheat expressed sequence tags (EST) databases based on the presence of amino acid sequence of Arabidopsis and rice MAPKs. Phylogenetic analyses of MAPKs from wheat, rice and Arabidopsis genomes have classified them into seven subgroups (A, B, C, D, E, F, and G). Using the available EST information as a source of expression data, the MAPK family genes from Triticum aestivum were detected in diverse tissues. Further expression analysis of the MAPKs in NCBI EST database revealed that their transcripts were most abundant in callus (20%), followed by leaf (12%) and inflorescence (12%). Most MAPK family genes showed some tissue specificity. Key words: wheat, MAPK, EST, phylogenetic analysis, profile
INTRODUCTION Mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant growth and development as well as biotic and abiotic stress responses (Ichimura 2002; Nakagami et al. 2005; Mishra et al. 2006). A classical MAPK pathway consists of three protein kinases, MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK. Upstream signals activate MAPKKKs (such as the Raf and Mos proteins), which then phosphorylate MAPKKs at a conserved S/T-X3-5S/T (X denotes any amino acid) motif. MAPKKs in turn activate a specific MAPK, through phosphorylation of the conserved threonine and tyrosine residues at a conserved T-X-Y (X denotes any amino acid) motif
Received 16 March, 2011
located in the activation loop (T-loop) between kinase subdomains VII and VIII. The specifically activated MAPKs phosphorylate various downstream targets and regulate the growth, development, and stress responses of organisms (Schaeffer and Weber 1999; Fu et al. 2002; Larade and Storey 2006). To date, the best understanding of MAPKs was in yeast, at least 5 out of 6 MAPK genes were characterized (Gustin et al. 1998). However, experimental evidence has shown that MAPK cascades in plants play much wider regulatory roles than those in animals and yeast (Mizoguchi et al. 1997; Fu et al. 2002). In plants, several dozens of MAPKs have been identified and isolated from Avena sativa (Huttly and Phillips 1995), Petunia juss (Decroocq-Ferrant et al. 1995), Zea mays (Lalle et al. 2005), Arabidopsis thaliana
Accepted 28 June, 2011
LIAN Wei-wei, Mobile: 15210842024, E-mail: [email protected]; Correspondence ZHAO Chang-ping, Tel: +86-10-51503712, E-mail: [email protected] * These authors contributed equally to this work. © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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(Mizoguchi et al. 1993), Hordeum vulgare (Knetsch et al. 1996), Nicotiana tabacum (Wilson et al. 1995), Oryza sativa (Fu et al. 2002), Chorispora bungeana (Zhang et al. 2006), respectively. During the past decade, incredible progress has been made towards the functional understanding of all genes in the model dicot Arabidopsis. In Arabidopsis genome, 20 MAPKs, 10 MAPKKs and 80 MAPKKKs were identified (Ichimura 2002). MAPK genes in rice (Oryza sativa) also were researched, and 17 members have been identified and analyzed (Reyna and Yang 2006). Wheat is one of the most important crops in the world. However, little is known about the MAPK genes and their regulatory roles in wheat. Up to date, only two TaMAPKs (WCK-1, FLRS) have been characterized in wheat (Takezawa 1999; Rudd et al. 2008). WCK-1 can be activated by fungal elicitors, which may involve Ca2+ in enhancing the MAP kinase signaling cascade in plants by controlling the levels of the MAP kinase transcripts (Takezawa 1999). The WCK-1 and FLRS are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola (Rudd et al. 2008). In this study, we conducted an in silico search of wheat EST databases to further identify members of the wheat MAPK gene family. A total of 15 genes were identified and among which 11 were novel. Meanwhile, a phylogenetic tree was constructed based on their homologous relationships. The MAPKs were grouped into 7 different subfamilies. Furthermore, a large group of Triticum aestivum ESTs was analyzed to gain insights into the MAPK family gene expression among different tissues.
RESULTS Collection of wheat MAPK genes Availability of wheat EST sequences has made it possible for the first time to identify the MAPK family members in this plant species (Gill et al. 2004). After carefully surveying the database, 15 putative genes were defined as wheat MAPKs by in silico analysis based on the presence of amino acid sequence of Arabidopsis and rice MAPKs (Table 1). Further, Fig. 1 showed that each of the wheat MAPK proteins carried a T[DE]Y motif between the subdomains VII and VIII that was necessary for activation of MAPK, which powerfully supported their reliability as the members of the MAPK family. Wheat MAPKs were named TaMAPKs in order to distinguish them from those having been cloned in wheat. In addition, TaMAPKs were numbered from 1 to 15 according to the numerical order they appeared on the phylogenetic tree.
Phylogenetic analysis of TaMAPKs Up to date, two TaMAPKs were identified (Takezawa 1999; Rudd et al. 2008). Manipulation of intracellular Ca2+ concentrations by treatment with calcium ionophore A23187 in the presence of high extracellular Ca2+ resulted in the induction of mRNA expression of WCK-1 (Takezawa 1999). FLRS and WCK-1 were associated with the resistance responses (Rudd et al. 2008). In Arabidopsis, MAPKs have been catego-
Table 1 List of MAPK genes in wheat grain Name TaMAPK1 TaMAPK2 TaMAPK3 * TaMAPK4 * TaMAPK5 * TaMAPK6 * TaMAPK7 * TaMAPK8 * TaMAPK9 * TaMAPK10 * TaMAPK11 TaMAPK12 * TaMAPK13 * TaMAPK14 TaMAPK15 *
UniGene number
Published MAPK
Accession number
T-Loop
AA length
Group
Ta.55196 Ta.23916 Ta.50588 Ta.54338 Ta.7205 Ta.14147 Ta.55675 Ta.4456 Ta.25413 Ta.13373 Ta.3070 Ta.18660 Ta.68359 Ta.6053 Ta.68224
WCK-1 FLRS
AF079318 AY173962
MAPK2B
DQ322666
MAPK1a
AY881102
TEY TEY TEY TEY TEY TEY TDY TDY TDY TDY TDY TDY TDY TDY TDY
369 403 387 376 369 369 548 598 696 613 549 494 490 578 498
B B A A C C D D D D E F F G G
Subcellular localization Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Periplasmic Periplasmic Periplasmic Periplasmicr Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic
, novel genes that we predicted according to silico cloning.
*
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
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Fig. 1 Protein sequence alignment of wheat MAPKs. Alignment was performed using the ClustalX2 program. The hosphorylation activation motif TxY(TEY and TDY) and kinase subdomains were indicated by box. It indicated that these TaMAPKs belonged to the MAPK gene families.
rized into four groups (A, B, C, and D) (Ichimura 2002). Using plant MAPKs from multiple plant species, phylogenetic analysis (Ligterink and Hirt 2001) suggests that plant MAPKs can be grouped into five groups (Fig. 2). Pairwise comparison and phylogenetic analysis of 20 AtMPKs and 17 OsMPKs indicated that these MAPKs can be divided into six groups (A, B, C, D, E, and F) (Reyna and Yang 2006). In order to investigate the phylogenetic relationships, the entire MAPK protein sequences were aligned by ClustalW and analyzed using MEGA4.0. In this study, the phylogenetic tree containing 20 AtMPKs, 15 TaMAPKs and 17 OsMPKs was used for further analysis. Here, these MAPKs can be divided into seven groups (A, B, C, D, E, F, and G). MAPKs in groups A, B, C contain the TEY motif in their activation site, whereas those in the D, E, F, and G groups contain the TDY activation motif (Fig. 1). Interestingly, wheat and rice contain more MAPKs with the TDY phosphorylation site than with the TEY motif. There are 9 TaMAPKs and 11 OsMAPKs containing the TDY motif whereas 6 TaMAPKs and 6 OsMAPKs belong to the TDY groups. In contrast, the Arabidopsis genome contains more MAPKs with the TEY phosphorylation site than with the TDY motif. 12 AtMAPKs have the TEY motif but only 8 AtMAPKs contain the TDY motif. Group A contains two wheat MAPKs (TaMAPK3 and TaMAPK4), which shows some similarity with 5 AtMPKs (AtMPK4, AtMPK12, AtMPK11, AtMPK5, and AtMPK13) and 2 OsMPKs (OsMPK2 and
OsMPK6). These MAPKs have been less well studied but appear to be involved in environmental stress responses and cell division. For example, disruption of the AtMPK4 gene that share high similarity with OsMPK2 and OsMPK6 by transposon insertion created a constitutive systemic acquired-resistance phenotype (Petersen et al. 2000). In the wild-type background, biochemical analysis using a MPK4-specific antibody revealed that both biotic and abiotic stresses induced activation of MPK4 (Ichimura et al. 2000; Desikan et al. 2001). Group B contains TaMAPK1 and TaMAPK2, which shares high similarity with OsMAPK5 and OsMAPK1, respectively, is known to be associated with a variety of environmental and hormonal responses. TaMAPK1 (WCK-1) can be activated by fungal elicitors and participate the defense response (Takezawa 1999). The first wheat MKP (Triticum turgidum L. subsp. durum) (accession no. EU502843), shares a highly homology with the WCK-1, interacts in vivo with TaMAPK1 and TaMAPK2 and controls their subcellular localization (Zaidi et al. 2010). Otherwise, the TaMAPK1 (WCK1) and TaMAPK2 (FLRS) are differentially regulated at multiple levels during compatible disease interactions with Mycosphaerella graminicola (Rudd et al. 2008). Group C includes two wheat MAPKs named TaMAPK5 and TaMAPK6 that shows high similarity with OsMPK4 and OsMPK3. Information on the group C MAPKs is limited, OsMPK4 (also known as OsMAPK4 or OsMSRMK3) has been shown to be tran-
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Fig. 2 Phylogenetic relationship of MAPKs. Phylogenetic analysis contains 20 AtMPKs, 15 TaMAPKs and 17 OsMPKs, and using MEGA4.0 to construct a neighbor joining (NJ) phylogenetic tree. These MAPKs can be divided into seven groups (A, B, C, D, E, F, and G). MAPKs in groups A, B, and C contain the TEY motif in their activation site, whereas those in the D, E, F, and G groups contain the TDY activation motif.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
scriptionally regulated by various abiotic stresses (Fu et al. 2002; Agrawal et al. 2003). Microarray analysis detected circadian-rhythm-regulated expression of AtMPK7 (Schaffer et al. 2001). Groups D, E, and F include MAPKs that have the TDY motif in their activation site and contain nine wheat MAPKs reported herein. Group D contains four wheat M A P K s ( Ta M A P K 7 , Ta M A P K 8 , Ta M A P K 9 , TaMAPK10). Five OsMPKs (OsMPK7, OsMPK8, OsMPK9, OsMPK10, and OsMPK11) and three AtMPKs (AtMPK18, AtMPK19, and AtMPK20) also belong to this group. The OsMPK8 (same as OsWJUMK1) gene is induced by various heavy metals, but its induction by pathogen attack has not been demonstrated previously (Fu et al. 2002). Group E contains a wheat MAPK named TaMAPK11 that shows high similarity with OsMPK14. OsMPK15 and AtMPK16 also belong to this group. Group F contains two TaMAPKs (TaMAPK12 and TaMAPK13) that shares high similarity with OsMPK17 and OsMPK16, respectively. Group G contains two wheat MAPKs named TaMAPK14 and TaMAPK15 that show high similarity with OsMPK12 and OsMPK13, respectively. Three Arabidopsis MAPKs (AtMPK9, AtMPK15, AtMPK8) also belong to this group. AtMPK8 and AtMPK9 possess a serine-rich and a glutamic-acid-rich region in their N-terminal regions, respectively (Ichimura 2002). The OsMPK12 gene (same as OsBWMK1) was shown to be transcriptionally induced by the rice blast fungus and wounding (He et al. 1999).
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Gene expression patterns of MAPKs To generate the patterns of expression of these MAPK family genes across various tissues and to assess the overall similarities and differences between transcriptomes of different tissues or organs, we performed a coordinated gene expression analysis of the wheat ESTs from different kinds of organs. The MAPK family genes were detected in all the ten types of plant tissues studied namely cell culture, flower, leaf, root, stem, callus, crown, inflorescence, sheath, and seed. Among ten types of plant tissues, transcripts of these MAPK family genes were shown to be most abundant in callus (20%), followed by leaf (12%) and inflorescence (12%) (Fig. 4). Some MAPK family genes were detected in other tissues as well, including sheath, root, cell culture, crown, stem, flower, and seed. To further characterize the pattern of expression of the single gene in different tissues, expression profiles of 15 MAPK
Structural comparison of wheat MAPKs TaMAPK proteins have a highly conserved kinase domain containing all fifteen subdomains that are characteristic of MAPKs. Like MAPKs in Arabidopsis and rice, the wheat MAPKs in groups A, B, and C have the TEY motif in the activation site. And a short C-terminus containing a common docking (CD) domain that consists of the sequence [LHY]Dxx[DE]EpxC (x represents any amino acid) serving as a docking site for MAPKKs, phosphatases, and protein substrates (Bardwell et al. 2003). Wheat MAPKs with the TDY motif do not have a CD domain but have a relatively long C terminal region (Fig. 3).
Fig. 3 Structural comparison of the 15 wheat MAPKs. The organization of the functional domains and motifs, including the phosphorylation motif (TxY) and CD domain of each MPAK, is shown in cartoon format. Scanning of the protein sequences for the presence of known motifs and domains was performed at PlantsP (http://plantsp.sdsc.edu/) (Gribskov et al. 2001).
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genes of wheat were examined by analyzing EST counts. Most of the MAPK family genes were detected in some tissues but not in others. TaMAPK13 exists in the whole plant, and has no expression profile database, so it has not been listed in the Fig. 5.
DISCUSSION Abiotic and biotic stress conditions such as cold, drought and pathogens attack seriously affect wheat growth and yield. To improve wheat tolerance to these
Fig. 4 Distribution of wheat MAPK family genes in various tissues.
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abiotic and biotic stresses, researches have been focused on the physiological and molecular mechanisms of wheat responses to these stresses. Isolation and identification of members of the MAPK family from wheat is of great importance for better understanding the molecular genetic basis for the wheat genetic improvement and providing the functional genetic resources required for transgenic research. Here, a total of 15 TaMAPKs were identified, which is fewer than those currently reported in Arabidopsis (23 members) and rice (17 members) (Ichimura 2002) because the whole genome of wheat is not sequenced and EST databases is limited. While, it is possible that more TaMAPKs may be identified by more wheat sequence (EST and genome) information in the future, it is likely that we have identified most, if not all, of the MAPKs in wheat. MAPKs phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK-mediated signaling is required both for patterning and for cell proliferation in the developing Drosophila wing (Marenda et al. 2006), which suggested the importance of subcellular localization of MAPKs. Based on prediction subcellular
Fig. 5 Tissue specific expression of the MAPK family genes.
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
localization of the MAPK proteins using the CELLO online software, we found TaMAPK7, TaMAPK8, TaMAPK9, and TaMAPK10 were mainly located in p e r i p l a s m i c , w h i l e o t h e r Ta M A P K s w e r e i n cytoplasmic, the mechanism of which was not yet known. In this study, according to phylogenetic analyses of MAPKs from wheat, rice and Arabidopsis, seven subgroups (A, B, C, D, E, F, and G) were found (Fig. 3), The A, B and C groups of the MAPKs contain the TEY phosphorylation site, whereas the D, E, F, and G groups have the TDY motif. Interestingly, there are fewer TaMAPKs in the A, B, and C groups (6 members) than in the D, E, F, and G groups (9 members). It is possible that this ratio (6 TEY TaMAPKs vs. 9 TDY TaMAPKs) may change with the discovery of more new TaMAPKs from the wheat genome. MAPKs within the same group might function similarly. For example, group A MAPKs were most frequently involved in environmental stress responses and cell division, whereas group B appeared to be associated with a variety of environmental and hormonal responses. Some of MAPKs in group C participated in various abiotic stresses regulation. On the other hand, group D, E, F, and G MAPKs were larger in size than other group MAPKs and little was known about the function of their long Cterminal extension. MAPKs ensure their signalling specificity of action by interacting with their substrates through common docking domains (CD domain). These docking domains lead the kinases to the correct substrates and enhance their fidelity and efficiency of action (Sharrocks et al. 2000), which was also observed in wheat groups A, B, and C MAPKs (Tanoue et al. 2000). However, the CD domain alone does not determine the docking specificity. Tanoue et al. (2001) found that a docking groove in the steric structure of MAPKs, which comprised the CD domain and the ED site, serving as a common docking region for various MAPK-interacting molecules. Docking sites are specific and modular, which are important for phosphorylation. Therefore, the existence of this conserved CD domain (i.e., docking sites) will help us understand the mechanism of interactions between MAPKs and a variety of other proteins in wheat. ESTs are created by partially sequenced gene tran-
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scripts and have proved valuable in molecular biology. ESTs have played an important role in gene discovery and in analyzing the patterns of gene expression (Fei et al. 2004). Previous study demonstrated that some MAPK family genes exhibited tissue specificity. The expression of RsMPK2 (a MAPK family gene from R. soongorica) was detected in vegetative (root, stem, leaf and callus) and reproductive (flower) organs, but the patterns of gene expression is diversity (Liu et al. 2010). A wheat MAPK gene, TaMAPK13, was expressed in all tissues abundantly, whereas the TaMAPK15, only expressed in inflorescence at a low level. To detect the pattern of expression of MAPK family genes, we analyzed the expression of these genes in various tissues. The result revealed that MAPK genes of wheat were strictly controlled in different organs and at different growth stages. These results will be helpful in studying biological functions of MAPK family genes.
CONCLUSION The present study has, for the first time, provided a comprehensive list of MAPKs present in wheat. In silico search of EST databases using BLASTP resulted in identification of 15 MAPK genes from wheat and among which 11 were new. These findings provide new insights into the diversity and structure of MARK genes in wheat and the biological function and evolution of these genes.
MATERIALS AND METHODS Identification of wheat TaMAPKs In order to isolate MAPK family genes in wheat, the amino acid sequence of 23 Arabidopsis and 17 rice MAPK genes were used as a query to search the database of UniGene (ftp://ftp.ncbi.nih.gov/repository/UniGene/ Triticum_aestivum/Ta.seq.all.gz), using the tBLASTn program at an e-value of 1e-3 to avoid false positives, then assemble the searched MAPK genes using the CAP3 sequence assembly program (http://pbil.univlyon1fr./cap3. php) (Huang and Madan 1999). ORFs were performed with the ORF Finder at NCBI (http://www.ncbi.nlm.nih.gov/gorf/ gorf. html).
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Conserved domain detection and subcellular localization prediction Programs SMART, MOTIF SCAN, and PLANTSP were used to detect conserved domains. In brief, the MAP kinase signature, PK domain, ATP-binding domain and so forth were found in a given protein sequence, which can be regarded as a candidate of wheat MAPK family. Prediction on subcellular localization of wheat MAPKs was carried out using the CELLO ver. 2.5 server (http://cello.life. nctu.edu.tw/).
Sequence alignment and phylogenetic analysis A multiple alignment analysis was performed with Clustal W. Phylogenetic trees were constructed using the neighborjoining (NJ) method and diagrams of phylogenetic trees were drawn with the help of MEGA4, the parameters were set to “multiple alignment gap opening penalty was 10, gap extension penalty was 0.2, delay divergent cutoff was 30%”.
Analysis of expression profile The expression profile was suggested by analyzing the EST counts based on UniGene (http://www.ncbi.nlm.nih. gov/UniGene/UGOrg.cgi?TAXID=3711).
Acknowledgements This work was supported by the Genetically Modified Organisms Breeding Major Projects, China (2008ZX08002002, 2008ZX08002-003, 2008ZX08002-004), the Beijing Technical Nova Project, China (2007B056, 2008B035), the Excellence Scholar Fostered Program of Beijing Government, China (20081D0200500050), the Beijing Natural Science Foundation of China (5102016) and Young Foundation Project of Beijing Academy of Agriculture and Forestry Scientific Research, China.
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Phylogenetic Analysis and Expression Patterns of the MAPK Gene Family in Wheat (Triticum aestivum L.)
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