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DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes logoLink to DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes
. 2010 Apr 15;17(3):139–153. doi: 10.1093/dnares/dsq011

In Silico Analysis Reveals 75 Members of Mitogen-Activated Protein Kinase Kinase Kinase Gene Family in Rice

Kudupudi Prabhakara Rao 1,‡, Tambi Richa 1,‡, Kundan Kumar 1,†, Badmi Raghuram 1, Alok Krishna Sinha 1,*
PMCID: PMC2885274  PMID: 20395279

Abstract

Mitogen-Activated Protein Kinase Kinase Kinases (MAPKKKs) are important components of MAPK cascades, which are universal signal transduction modules and play important role in plant growth and development. In the sequenced Arabidopsis genome 80 MAPKKKs were identified and currently being analysed for its role in different stress. In rice, economically important monocot cereal crop only five MAPKKKs were identified so far. In this study using computational analysis of sequenced rice genome we have identified 75 MAPKKKs. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analyses of MAPKKKs from rice and Arabidopsis have classified them into three subgroups, which include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Further expression analysis of the MAPKKKs in MPSS database revealed that their transcripts were differentially regulated in various stress and tissue-specific libraries.

Keywords: MAPK cascade, MAPKKK, gene family, rice

1. Introduction

Mitogen-Activated Protein Kinase (MAPK) cascade plays an important role in plant growth and development, transferring the extracellular stimuli into intracellular response. MAPK cascades are evolutionarily conserved signalling modules in eukaryotes including animals, yeasts and plants.1,2 MAPK cascades are composed of three protein kinases: MAPKs, MAPK Kinases (MAPKKs/MKKs) and MAPKK Kinases (MAPKKKs/MEKKs). MAPKs are activated when both tyrosine and threonine residues in the TXY motif are phosphorylated by MAPKKs. MAPKKs are activated when serine and serine/threonine residues in the S/TXXXXXS/T motif are phosphorylated by MAPKKKs.3

By sequence comparison and signature motif searches, putative orthologue of MAPK cascade members have been identified in rice, Medicago sativa, Zea mays, tobacco and tomato. Southern blot analysis have also revealed that genomes of monocotyledonous plants such as rice and maize possesses sequences that are homologous to the NPKl gene (MAPKKK related gene in tobacco), an indication that NPKl-related genes are present in a number of plant species.4 During the past decade, incredible progress has been made towards the functional understanding of all genes in the model dicot Arabidopsis. In the sequenced Arabidopsis genome, 20 MAPKs, 10 MAPKKs and 80 MAPKKKs were identified.5,6 However, little is known about the MAPK gene family and their function and regulation in rice (Oryza sativa) and other economically important cereal crops.7 After the completion rice genome project 16 MAPKs and 8 MAPKKs are reported in rice.8 However, reports on the presence of MAPKKKs in rice have been very slow and so far a total of only five MAPKKKs have been reported. MAPKKK, which gets activated by upstream signals, forms a very important component of MAPK cascade. In plants the roles MAPKKK has been identified in various stresses,9,10 plant cytokinesis,11,12 ethylene signalling,13 innate immunity14 and defence responses,15,16,17 among many others.

However, the poor information about MAPKKK gene family in rice is proving to be bottleneck in elucidating MAPK cascade in this very important monocot crop. In the present study, an in silico search of rice genome databases was conducted to identify members of the rice MAPKKK gene family. A total of 75 genes were identified and among which 70 were novel. A phylogenetic tree was constructed and MAPKKKs were grouped into three different subfamilies. Conserved consensus motifs were analysed in all the subfamilies to support their association. To further validate the findings, expression analysis of all the 75 MAPKKKs in different stresses and tissue-specific libraries were carried out using MPSS database.

2. Materials and methods

2.1. Sequence and database search for OsMAPKKKs

In an attempt to obtain all the MAPKKKs, rice protein sequences available in Rice Genome Annotation Project (ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/ O_sativa/annotation_dbs/pseudomolecules/version_6.0) were downloaded to construct a local protein database. It comprised of 67393 sequences. This database was searched with MAPKKK query sequences downloaded from NCBI (ncbi.nlm.nih.gov), TAIR (arabidopsis.org), Royal Holloway University of London (www.rhul.ac.uk./Biological-Sciences/AcademicStaff/Bogre/mapkkk.htm), MIPS (mips.gsf.de), Uniprot (uniprot.org), BrassicaDB (brassica.bbsrc.ac.uk/BrassicaDB), MPSS (mpss.udel.edu/rice) and Maizegdb (maizegdb.org). The query consisted of 100 sequences from 13 different plant species which included Arabidopsis thaliana, Brassica napus, Cucumis melo, Cucumis sativus, Lotus japonicus, Lycopersicon esculentum, M. sativa, Nicotiana tabacum, O. sativa, Solanum chacoense, Triticum aestivum, Vitis vinifera and Z. mays. The search was carried out using BLASTP and 50% identity was taken as the threshold for the sequences obtained from BLAST analysis. Further all the MAPKKK sequences were aligned using CLUSTAL 2.0.3 (http://www.ebi.ac.uk/clustalw/) multiple sequence alignment and used to construct HMM profile. Using HMMER 2.3.2 (ftp://ftp.genetics.wustl.edu/pub/eddy/hmmer), the local protein database was searched on the basis of the HMM profile. HMMER and BLAST hits were compared and parsed by manual editing as well as using small PERL scripts. In addition self BLAST of the sequences was carried out to remove the redundancy and then they were analysed for the presence of domains. Subcellular localization prediction of each of the rice MAPKK kinases was carried out using the CELLO v2.5 server (http://cello.life.nctu.edu.tw/).

2.2. Phylogenetic tree construction

The Kinase domain of all the sequences (including both query and hits obtained) were analysed using ScanProsite program from Expasy server (au.expasy.org/prosite/). All the kinase domains were aligned using ClustalW. To investigate the evolutionary relationship among MAPKKK proteins, a phylogenetic tree was constructed by employing the minimal evolution (ME) method and the neighbour-joining (NJ) method wrapped in MEGA4 software suite.

2.3. Multiple alignment and conserved signature detection

Multiple sequence alignments of related proteins belonging to each group from both Arabidopsis and rice were performed using Multalin, which creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments (http://bioinfo.genotoul.fr/multalin/multalin). Programs INTERPROSCAN, SMART and MOTIF SCAN were employed to detect conserved domains.

2.4. Analysis of MPSS database for expression profiles

To gain insight into expression profiles of OsMAPKKK members in O. sativa in different stress and tissues, the MPSS database (http://mpss.udel.edu/rice/) was searched (opting 20-nt signature sequences) using the locus ID given in the TIGR database. The data thus obtained have been analysed and grouped based on stress and tissue specificity.

3. Results and discussion

3.1. In silico search and identification of novel MAPKKK genes

Availability of complete rice genome sequences18 has made it possible for the first time to identify all the MAPKKK family members in this plant species. In order to identify the MAPKKK genes, 100 query sequences of MAPKKKs from different plant species were analysed by BLASTP against 67393 sequences of the local protein database of rice from RGP, which resulted in 122 hits as subject sequences. These hits were passed through several layers of filters which primarily included 50% identity with the query sequence which further reduced to 107 hits as subject sequences. This list included all the five MAPKKKs from rice, which were already reported in NCBI and MPSS database. Simultaneously an HMM profile (HMMER version 2.3.2) was created with the 100 query sequences and a profile search was carried out against the local rice proteome database which resulted in 1574 hits. After the comparison of the sequences obtained from BLAST hits and HMM searches, the number of MAPKKKs was further reduced to 102. A self BLAST of these sequences followed by manual editing to remove the redundancy finally resulted in identification of 75 MAPKKK genes. This analysis has revealed that the O. sativa genome has 75 putative MAPKKK genes including five already known MAPKKKs. However, in Arabidopsis 80 putative MAPKKKs were predicted.6 Most of the MAPKKKs existence was supported by EST hits and full-length cDNA sequences from KOME or GenBank database (Table 1) except in 14 MAPKKKs where no such data were available. It indicates either all 14 are not expressed in all the conditions used for analysis or that they are expressed in very low quantity, which cannot be detected. Expression of other members was found in either of the two conditions mentioned above. MPSS database measures the absolute expression level of most genes in the sample and provide information about potentially novel transcripts.19 Out of the 14 MAPKKKs which were not reported in EST or cDNA database, eight of them were represented in MPSS database with their expression levels in different tissues and stresses and in the remaining six MAPKKKs, five were without any expression in the tested conditions additionally OsMAPKKK53 was not represented in MPSS database. Since there was no standard nomenclature followed for MAPKKKs neither in Arabidopsis nor in rice, we named the MAPKKKs sequentially based on the HMM search output. All the 75 MAPKKKs were having conserved protein kinase domain which is backbone for MAPK family. The predicted protein localization of most of the MAPKKKs varied from cytoplasm, mitochondria, chloroplast to nucleus except in MAPKKK33 and MAPKKK47 where it was present in cytoskeleton and peroxisomes, respectively.

Table 1.

List of MAPKKKs from rice

Sl. No. MAPKKKs Score TIGR/MSU ID Amino acid length Total number of mapped ESTs ID's of cDNAs Subcellular localization TIGR predicted function (from MPSS Db.)
1 MAPKKK1 529.3 LOC_Os03g06410 1017 74 AY167575 AK111595 Nuclear EDR1, putative, expressed
2 MAPKKK2 522 LOC_Os10g29540 972 30 AK121718 Nuclear EDR1, putative, expressed
3 MAPKKK3* 515.1 LOC_Os02g32610 781 25 CT835420 Chloroplast Protein kinase domain containing protein, expressed
4 MAPKKK4 513.1 LOC_Os02g12810 864 31 N/A Chloroplast MAP3K delta-1 protein kinase, putative, expressed
5 MAPKKK5 512.9 LOC_Os12g37570 758 24 N/A Nuclear ATP binding protein, putative, expressed
6 MAPKKK6 497.4 LOC_Os02g50970 1111 42 AK102767 Nuclear Protein kinase domain containing protein, expressed
7 MAPKKK7 491.2 LOC_Os06g12590 1078 17 AK099500 AK105681 Plastid ATP binding protein, putative, expressed
8 MAPKKK8 489.3 LOC_Os11g10100 653 35 N/A Nuclear Mitogen-activated kinase kinase kinase alpha, putative, expressed
9 MAPKKK9 489.1 LOC_Os02g44642 894 22 AK073040 Nuclear YDA, putative, expressed
10 MAPKKK10 485.3 LOC_Os04g47240 894 38 N/A Nuclear YDA, putative, expressed
11 MAPKKK11# 482.4 LOC_Os07g02 780 753 7 AK069889 Chloroplast MAPKKK5, putative, expressed
12 MAPKKK12 482 LOC_Os09g39320 1220 6 N/A Cytoplasm CTR1-like protein kinase, putative, expressed
13 MAPKKK13 479.6 LOC_Os09g21510 848 0 N/A Nuclear Mitogen-activated protein kinase kinase kinase 2, putative, expressed
14 MAPKKK14 464.8 LOC_Os04g52 140 778 48 AK120898 Chloroplast Protein kinase domain containing protein, expressed
15 MAPKKK15 463.4 LOC_Os08g32600 690 9 AK103087 Nuclear Protein kinase domain containing protein, expressed
16 MAPKKK16 462.9 LOC_Os04g35700 708 38 AK061622 AK068725 Nuclear Mitogen-activated kinase kinase kinase alpha, putative, expressed
17 MAPKKK17 461.8 LOC_Os09g37230 603 57 AK072690 Cytoplasmic ATP binding protein, putative, expressed
18 MAPKKK18 461.6 LOC_Os03g55560 777 112 N/A Chloroplast MAPKKK5, putative, expressed
19 MAPKKK19 457.5 LOC_Os02g35010 690 9 AK287889 Mitochondria MAP3KA, putative, expressed
20 MAPKKK20 453.9 LOC_Os07g38530 704 32 DQ837532 AK100426 AF080436 Nuclear Protein kinase domain containing protein, expressed
21 MAPKKK21 446.7 LOC_Os07g25680 1219 6 AK241123 Nuclear Protein kinase domain containing protein, expressed
22 MAPKKK22* 443.1 LOC_Os03g49640 654 15 N/A Nuclear Mitogen-activated protein kinase, putative, expressed
23 MAPKKK23 440.3 LOC_Os12g40279 4262 1 N/A Plastid Protein kinase domain containing protein, expressed
24 MAPKKK24# 440.1 LOC_Os04g56530 1357 54 AK099839 Cytoplasm MAPKKK7, putative, expressed
25 MAPKKK25 438 LOC_Os02g38080 352 4 N/A Cytoplasm Mitogen-activated protein kinase kinase kinase 7, putative, expressed
26 MAPKKK26 437.6 LOC_Os07g29330 439 27 AK066198 Cytoplasm Serine/threonine-protein kinase CTR1, putative, expressed
27 MAPKKK27 436.5 LOC_Os03g43760 379 98 AK243690 Cytoplasm ATP binding protein, putative, expressed
28 MAPKKK28 436.1 LOC_Os03g15570 597 51 AK242766 CT835152 CT835203 Nuclear Mitogen-activated protein kinase 1, putative, expressed
29 MAPKKK29 434.4 LOC_Os02g45130 612 42 AK073845 Chloroplast WNK6, putative, expressed
30 MAPKKK30 434.3 LOC_Os02g02780 583 49 AK060220 AK103704 Cytoplasm ATP binding protein, putative, expressed
31 MAPKKK31 429.9 LOC_Os01g45380 388 1 AK108130 Cytoplasm ATP binding protein, putative, expressed
32 MAPKKK32 426.2 LOC_Os08g12750 418 103 AY156510 AK099003 AK111601 AK111800 AK121704 AY646225 Cytoplasm Serine/threonine-protein kinase, putative, expressed
33 MAPKKK33 423.9 LOC_Os02g07790 421 83 AY156512 AK101327 Cytoskeleton Serine/threonine-protein kinase CTR1, putative, expressed
34 MAPKKK34 423.4 LOC_Os05g50190 381 23 AY224453 AK107217 Nuclear ATP binding protein, putative, expressed
35 MAPKKK35 419.3 LOC_Os02g54510 1083 2 N/A Nuclear Protein kinase domain containing protein, expressed
36 MAPKKK36 418.6 LOC_Os05g01780 621 40 AK070061 Nuclear ZIK1 protein, putative, expressed
37 MAPKKK37 418.5 LOC_Os04g51950 422 54 AY156511 AK111698 Cytoplasm Serine/threonine-protein kinase, putative, expressed
38 MAPKKK38 416.7 LOC_Os06g45300 428 49 AY224431 AK112024 Cytoplasm Serine/threonine-protein kinase, putative, expressed
39 MAPKKK39 416.2 LOC_Os06g08280 1273 27 AK067771 Nuclear Protein kinase domain containing protein, expressed
40 MAPKKK40 415.7 LOC_Os01g48330 801 66 AK070808 AK102209 AK111983 Nuclear ATP binding protein, putative, expressed
41 MAPKKK41 408.5 LOC_Os06g43840 1112 49 AK243413 Nuclear Protein kinase domain containing protein, expressed
42 MAPKKK42 405.8 LOC_Os03g60150 383 67 CT830832 Cytoplasm Tyrosine-protein kinase 2, putative, expressed
43 MAPKKK43 400 LOC_Os06g50920 564 16 AK073747 Cytoplasm ATP binding protein, putative, expressed
44 MAPKKK44 399.5 LOC_Os02g14530 790 50 AK111618 Nuclear ATP binding protein, putative, expressed
45 MAPKKK45 399 LOC_Os06g43030 398 0 N/A Chloroplast HT1 protein kinase, putative, expressed
46 MAPKKK46 394.2 LOC_Os11g06140 439 3 AK070490 Nucleus Serine/threonine-protein kinase WNK4, putative, expressed
47 MAPKKK47 380.6 LOC_Os07g08750 601 37 AK060552 AK100930 Peroxisome WNK1, putative, expressed
48 MAPKKK48 371.7 LOC_Os01g01740 376 32 AK059460 Nuclear HT1 protein kinase, putative, expressed
49 MAPKKK49 366.1 LOC_Os05g44290 604 3 N/A Nuclear Protein kinase, putative, expressed
50 MAPKKK50 363.3 LOC_Os12g02250 619 48 AK067447 AK072172 Nuclear Mitogen-activated protein kinase, putative, expressed
51 MAPKKK51 361.1 LOC_Os01g54350 637 38 AK102467 Nuclear Protein kinase, putative, expressed
52 MAPKKK52 357.4 LOC_Os12g06490 418 71 AK062812 AK073772 Cytoplasm Serine/threonine-protein kinase WNK3, putative, expressed
53 MAPKKK53 355.8 LOC_Os11g02305 622 0 N/A Nuclear –
54 MAPKKK54 355.5 LOC_Os03g28300 859 63 CT828499 Nuclear ATP binding protein, putative, expressed
55 MAPKKK55 354.1 LOC_Os01g50400 418 0 N/A Chloroplast Mitogen-activated protein kinase kinase kinase 1, putative, expressed
56 MAPKKK56 349.8 LOC_Os05g01780 621 20 AY336987 Mitochondria ZIK1 protein, putative, expressed
57 MAPKKK57# 349.4 LOC_Os05g46750 591 0 N/A Chloroplast MAPKKK16, putative, expressed
58 MAPKKK58 348.8 LOC_Os03g39150 351 0 N/A Cytoplasm Serine/threonine-protein kinase CTR1, putative
59 MAPKKK59 346.4 LOC_Os12g41260 400 1 AK109696 Cytoplasm ATMRK1, putative, expressed
60 MAPKKK60 341.8 LOC_Os03g53410 407 11 N/A Chloroplast ATMRK1, putative, expressed
61 MAPKKK61 334.7 LOC_Os01g10450 563 25 AK069537 Nuclear Protein kinase, putative, expressed
62 MAPKKK62 329.7 LOC_Os01g50420 541 0 N/A Mitochondria Mitogen-activated protein kinase kinase kinase 2, putative, expressed
63 MAPKKK63 328.8 LOC_Os01g50370 484 0 N/A Chloroplast Mitogen-activated protein kinase kinase kinase 1, putative, expressed
64 MAPKKK64 325.1 LOC_Os07g39520 327 42 CT832200 AK072014 AK072176 AK105268 Mitochondria Serine/threonine-protein kinase WNK2, putative, expressed
65 MAPKKK65 324 LOC_Os07g43900 321 12 AK241519 Cytoplasmic Mitogen-activated protein kinase kinase kinase 10, putative, expressed
66 MAPKKK66 320.8 LOC_Os10g04010 525 0 N/A Chloroplast Mitogen-activated protein kinase kinase kinase 1, putative
67 MAPKKK67 318.7 LOC_Os10g04000 526 0 N/A Cytoplasm Mitogen-activated protein kinase kinase kinase 1, putative
68 MAPKKK68 315.8 LOC_Os12g30570 384 0 N/A Nuclear ATMRK1, putative
69 MAPKKK69 305.8 LOC_Os05g46760 441 8 AK105946 Chloroplast Mitogen-activated protein kinase kinase kinase 1, putative, expressed
70 MAPKKK70 300.2 LOC_Os01g50410 451 0 N/A Mitochondria Mitogen-activated protein kinase kinase kinase 1, putative, expressed
71 MAPKKK71 278.4 LOC_Os02g21700 413 0 N/A Cytoplasm Mitogen-activated protein kinase kinase kinase 2, putative, expressed
72 MAPKKK72 265.4 LOC_Os01g54480 468 29 AK104674 Mitochondria Serine/threonine-protein kinase TNNI3K, putative, expressed
73 MAPKKK73 258.8 LOC_Os03g18170 511 0 N/A Chloroplast Mitogen-activated protein kinase kinase kinase 1, putative, expressed
74 MAPKKK74 225 LOC_Os01g66860 500 13 AK070097 Chloroplast Ankyrin-kinase, putative, expressed
75 MAPKKK75 212.5 LOC_Os02g39560 502 13 AK071922 AK099756 Chloroplast Non-receptor tyrosine kinase spore lysis A, putative, expressed
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*From NCBI database (MAPKKK3 and MAPKKK22).

#From MPSS database (MAPKKK11, MAPKKK24 and MAPKKK57).

3.2. Phylogenetic analysis of MAPKKKS

The family of MAPKKKs forms the largest group of MAPK pathway components. Arabidopsis contains 80 MAPKKKs,6 which can be subdivided into three major subtypes, Raf, MEKK and ZIK.20 Among these two groups, MEKK group is most similar to animal MEKKs and yeast MAPKKKs. In total, it consists of 21 MEKK-like and 11 ZIK kinases along with Arabidopsis ANP1-3 (Arabidopsis NPK1-like protein kinases). The other group consists of 48 genes encoding Raf-like protein kinases including Arabidopsis CTR1 and EDR1.6 To further characterize the MAPKKKs from rice and to evaluate the phylogenetic relationships with Arabidopsis MAPKKKs, the kinase domains of rice and Arabidopsis were aligned using ClustalW and analysed using MEGA4. Phylogenetic tree was constructed by employing the NJ method (Fig. 1) and ME method have shown similar topologies, with only minor modifications at deep nodes. On the basis of phylogenetic analysis, MAPKKKs in rice were classified in to three categories, which include Raf, ZIK and MEKK subfamilies. There were 43 MAPKKKs from rice and 48 from Arabidopsis grouped under Raf subfamily, 22 MAPKKKs from rice and 21 from Arabidopsis were grouped in to MEKK subfamily where as only 10 MAPKKKs from rice and 11 from Arabidopsis were grouped under ZIK family. As the results indicate, rice and Arabidopsis show similarity in the number of MAPKKKs in each subgroup.

Figure 1.

Figure 1

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Phylogenetic tree and domain organization of MAPKKKs from rice and Arabidopsis. Kinase domains of putative MAPKKKs from Arabidopsis and rice were aligned using CLUSTALW program. The NJ phylogenetic tree was created using MEGA4 software suite. Bootstrap value above 50% was shown. The domain organization was depicted on the right, gaps in lines were introduced while depicting the protein sequences of bigger size for convenience. Scanning of the protein sequences for the presence of known motifs and domains was performed using PlantsP. To identify the species of origin for each MAPKKK, a species acronym is included before the protein name: At, Arabidopsis thaliana; Os, Oryza sativa.

Domain architecture of MAPKKKs from Arabidopsis and rice revealed that most of the Raf family proteins have a C-terminal kinase domain and a long N-terminal regulatory domain. In contrast, majority of the ZIK family members have N-terminal kinase domain whereas members of MEKK family has less conserved protein structure with kinase domain located either at N- or C-terminal or central part of the protein. Bipartite NLS and transmembrane regions are distributed across the members of all the subfamilies whereas extensin like region is observed in Raf and MEKK members. Ubiquitin-interaction motif and ACT domain which is known to play a role in the regulation of a wide range of metabolic enzymes by responding to amino acid concentration are present only in the members of Raf family from rice and Arabidopsis (Fig. 1).

Extrapolating the phylogenetic tree shown in Fig. 1 along with 14 additional sequences from 11 different plants revealed that most of the known MAPKKK from other plants are grouped with MEKK subfamily (Supplementary Fig. S1). Comparing MPK and MKK gene families in rice, Arabidopsis and Populus genomes it was reported that the recent duplication events in amplifying the respective gene families are more evident in eudicots compared with monocot rice.8 We also observed comparing the MAPKKK gene family between rice and Arabidopsis that the eudicot has undergone more recent duplication events, more so in the Raf subfamily (Supplementary Fig. 1).

In silico localization of MAPKKKs on chromosomes indicated that all the 75 MAPKKKs are distributed on all 12 chromosomes of rice and half of them were present in first three chromosomes, which include chromosome 1, 2 and 3. Among all, chromosomes 8 and 11 were sharing only two MAPKKKs each, whereas chromosome 2 was having as many as 13 MAPKKKs (Fig. 2). Although similar analysis for other two components of MAPK cascade namely, MAPKK/MKK and MAPK/MPK revealed that eight MKKs are present in four chromosomes and 16 MPKs are distributed on seven chromosomes.

Figure 2.

Figure 2

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Graphical (scaled) representation of location of MAPK, MAPKK and MAPKKK genes on rice chromosomes.

3.3. Analysis of conserved motifs among MAPKKKs

The pattern of amino acid residues found in many subdomains is conserved among the family members. All the rice MAPKKKs that were grouped under Raf, ZIK and MEKK subfamilies were further analysed for the presence of specific signatures. Raf subfamily consists of the largest number of MAPKKKs in both Arabidopsis and rice. Experimental data from Drosophila and Caenorhabditis elegans have provided much evidence that the Rafs sensu stricto stimulate MAP2K and MAPK activation.21 Human Rafs are involved in a signalling network that controls cell proliferation, cell differentiation and apoptosis. Many of their effects are transmitted through the ERK/MAPK pathway.22,23

Raf family has a conserved signature in its kinase domain across the members. Analysis of the rice MAPKKKs along with the Arabidopsis for Raf specific signature GTXX (W/Y) MAPE was carried out by multiple alignments of kinase domains. The data revealed the presence of signature in all the members of Raf family in rice (Fig. 3) and strongly supported their identity as members of Raf subfamily. Around 43 MAPKKKs were grouped under Raf subfamily in rice where as in Arabidopsis this number is higher and consists of 48 members. The kinase domain of human B-Raf is a strong activator of MEK and has a high affinity for MEK.24,25 In Arabidopsis, members of Raf subfamily CTR1 and EDR1 act as negative regulators in ethylene signalling13 and in response to powdery mildew attack,26 respectively. Further CTR1 has been found to interact with the histidine kinase domain of ETR1 and the ethylene response sensor (ERS1) in vitro.27

Figure 3.

Figure 3

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Alignment of MAPKKKs of Raf subfamily from rice and Arabidopsis. The highlighted part shows the conserved signature motif.

The ZIK subfamily consists of 10 putative MAPKKKs in rice and 11 in Arabidopsis. Recently, the Arabidopsis ZIK protein WNK1 (At3g04910) was demonstrated to phosphorylate a protein involved in the control of circadian rhythms,28 suggesting a function different from that of other MAPKKKs. The characteristic feature of this family consists of a conserved signature GTPEFMAPE (L/V/M) (Y/F/L) across the members. Rice putative MAPKKKs were analysed for the presence of the above signature, which reassured that 10 MAPKKKs out of 75 have ZIK specific signatures (Fig. 4) and eventually grouped under this family.

Figure 4.

Figure 4

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Alignment of MAPKKKs of ZIK subfamily from rice and Arabidopsis. The highlighted part shows the conserved signature motif.

Among the three families MEKK subfamily is relatively well characterized. NPK1 gene from tobacco whose role in cytokinesis have been established,11,29 ScFRK2, a MAPKKK from S. chacoense involved in fertilization and embryogenesis,30 OMTK1, MAPKKK from M. sativa which channels oxidative stress signalling31 were some of the members of MEKK subfamily. Further ANP1 in Arabidopsis was found responsive to oxidative stress and is involved in negative regulation of auxin signal transduction pathway.32,33 It was also reported that Arabidopsis ANP1, ANP2, ANP3 are involved in plant cytokinesis11,34 and MAPKKKα in relation to defence response.15 Role of YODA a MAPKKK from Arabidopsis was characterized in stomatal development35 with its targeting downstream MAPKKs.36 Another member of this family MEKK1 functions in integrating ROS homeostasis with plant development and hormone signalling.37,38 Twenty-two MAPKKKs from rice and 21 from Arabidopsis belong to this subfamily. Relationship analysis based on the amino acid sequences of the protein kinase catalytic domain shows that the sequence, G (T/S) PX (F/Y/W) MAPEV forms a conserved signature of this family (Fig. 5). Presence of this signature in 18 putative rice MAPKKKs further confirmed their association with MEKK family. Moreover analysis of the transcript abundance of MAPKKK subfamilies from rice in different tissue and stress specific libraries revealed that they are differentially expressed and regulated.

Figure 5.

Figure 5

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Alignment of MAPKKKs of MEKK subfamily from rice and Arabidopsis. The highlighted part shows the conserved signature motif.

3.4. In silico analysis of expression of MAPKKKs based on MPSS database

MPSS is a valuable tool to have an insight into gene expression.39 It has been used previously for genome-level expression analysis in several systems including Arabidopsis.40 To extract information about the relative abundance of transcripts of O. sativa MAPKKK members, we have carried out the analysis in the available MPSS database (http://mpss.udel.edu/rice/). This database is derived from the TIGR O. sativa genome sequence and the search has been performed employing the 20 nucleotides long signatures in tissue-specific and stress-related libraries. Our analysis has revealed that among the different tissue-specific libraries, maximum numbers of MAPKKKs were expressed and their transcript abundance was found in crown vegetative meristematic tissue (NME), ovary and mature stigma (NOS) tissue libraries. Libraries of salinity and cold stress in rice roots have shown maximum transcript abundance of OsMAPKKK64 which is a member of ZIK subfamily (Fig. 6) and with drought stress elevated transcripts of OsMAPKKK28 was observed (Fig. 7). OsMAPKKK28 is a member of MEKK subfamily and an orthologue of AtMEKK1 from Arabidopsis. Similarly in leaves highest Transcripts Per Million (TPM) of OsMAPKKK4 a member of Raf subfamily in salinity stress (Fig. 8), OsMAPKKK63 in cold, OsMAPKKK8 in drought, all members of MEKK subfamily, were observed. This suggested the involvement of MAPKKK members from all the subfamilies and more precisely MEKK subfamily in the regulation of abiotic stress. These observations further strengthen their prediction and the differential regulation in various stress conditions suggests their active participation in stress signalling.

Figure 6.

Figure 6

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Transcript abundance of MAPKKK members of ZIK subfamily in salinity, drought, cold and tissue-specific libraries from MPSS database along with their dendrogram. Different libraries in MPSS database were analysed for the expression level of MAPKs. NYR, 14 days young roots; NSR, 14 days young roots stressed in 250 mM NaCl for 24 h; NDR, 14 days young roots stressed in drought for 5 days; NCR, 14 days young roots stressed in 4°C cold for 24 h; NYL, 14 days young leaves; NSL, 14 days young leaves stressed in 250 mM NaCl for 24 h; NDL, 14 days young leaves stressed in drought for 5 days; NCL, 14 days young leaves stressed in 4°C Cold for 24 h; NRA, mature roots (60 days) replicate A; NST, mature stem (60 days); NLA, mature leaves (60 days) replicate A; NME, Crown vegetative meristematic tissue (60 days); NPO, mature pollen; NOS, ovary and mature stigma; NIP, Immature panicle; NGS, 3 days germinating seed.

Figure 7.

Figure 7

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Transcript abundance of MAPKKK members of MEKK subfamily in salinity, drought, cold and tissue-specific libraries from MPSS database along with their dendrogram. Different libraries in MPSS database were analysed for the expression level of MAPKs. NYR, 14 days young roots; NSR, 14 days young roots stressed in 250 mM NaCl for 24 h; NDR, 14 days young roots stressed in drought for 5 days; NCR, 14 days young roots stressed in 4°C cold for 24 h; NYL, 14 days young leaves; NSL, 14 days young leaves stressed in 250 mM NaCl for 24 h; NDL, 14 days young leaves stressed in drought for 5 days; NCL, 14 days young leaves stressed in 4°C cold for 24 h; NRA, mature roots (60 days) replicate A; NST, mature stem (60 days); NLA, mature leaves (60 days) replicate A; NME, crown vegetative meristematic tissue (60 days); NPO, mature pollen; NOS, ovary and mature stigma; NIP, immature panicle; NGS, 3 days germinating seed.

Figure 8.

Figure 8

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Transcript abundance of MAPKKK members of Raf subfamily in salinity, drought, cold and tissue-specific libraries from MPSS database along with their dendrogram. Different libraries in MPSS database were analysed for the expression level of MAPKs. NYR, 14 days young roots; NSR, 14 days young roots stressed in 250 mM NaCl for 24 h; NDR, 14 days young roots stressed in drought for 5 days; NCR, 14 days young roots stressed in 4°C cold for 24 h; NYL, 14 days young leaves; NSL, 14 days young leaves stressed in 250 mM NaCl for 24 h; NDL, 14 days young leaves stressed in drought for 5 days; NCL, 14 days young leaves stressed in 4°C cold for 24 h; NRA, mature roots (60 days) replicate A; NST, mature stem (60 days); NLA, mature leaves (60 days) replicate A; NME, crown vegetative meristematic tissue (60 days); NPO, mature pollen; NOS, ovary and mature stigma; NIP, immature panicle; NGS, 3 days germinating seed.

3.5. Conclusion

The present study has provided the full list of MAPKKKs present in rice for the first time. In silico search of various rice protein databases using BLASTP and HMM profile resulted in identification of 75 MAPKKK genes from rice among these 70 were novel. EST hits and full-length cDNA sequences (from KOME or Genbank database) of 75 MAPKKKs supported their existence. Phylogenetic analysis of MAPKKKs from rice and Arabidopsis has classified them in to three subgroups that include Raf, ZIK and MEKK. Conserved motifs in the deduced amino acid sequences of rice MAPKKKs strongly supported their identity as members of Raf, ZIK and MEKK subfamilies. Expression analysis of the MAPKKKs in MPSS database revealed that maximum number of MAPKKK transcripts was represented in crown vegetative meristematic tissue (NME), ovary and mature stigma (NOS) tissue libraries. Libraries of salinity and cold stress in rice roots have shown maximum transcript abundance of OsMAPKKK64 which is a member of ZIK subfamily and with drought stress elevated transcripts of OsMAPKKK28 was observed. Similarly in leaves highest TPM of OsMAPKKK4 a member of Raf subfamily in salinity stress, OsMAPKKK63 in cold, OsMAPKKK8 in drought, which are members of MEKK subfamily were observed. The information generated will be very significant for further investigating the regulation mechanism of MAPKKKs and eventually MAPK cascade in response to extracellular stimuli and their central roles in various biological functions. Additionally, the information generated will serve the purpose in elucidating still very poorly characterized MAP kinase cascade in plants in general and in rice in particular.

Supplementary Data

Supplementary Data are available at www.dnaresearch.oxfordjournals.org.

Funding

The work is financially supported by the core grant of National Institute of Plant Genome Research, New Delhi, India.

Supplementary Material

[Supplementary Data]
dsq011_index.html (825B, html)

Acknowledgements

K.P.R. and K.K. acknowledge the fellowship provided by University Grant Commission, India, while B.R. thanks Department of Biotechnology, India for fellowship.

Footnotes

Edited by Kazuo Shinozaki

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Associated Data

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Supplementary Materials

[Supplementary Data]
dsq011_index.html (825B, html)
dsq011_1.pdf (963.2KB, pdf)
dsq011_2.pdf (38.7KB, pdf)

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