title: Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).
abstract: BACKGROUND
  Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses.
  
  
  RESULTS
  We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization.
  
  
  CONCLUSIONS
  We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China.
authors.forename: Wanwan
authors.initials: W
authors.lastname: Liang
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Bao-Jun
authors.initials: BJ
authors.lastname: Yu
authors.forename: Zili
authors.initials: Z
authors.lastname: Zhou
authors.forename: Cui
authors.initials: C
authors.lastname: Li
authors.forename: Ming
authors.initials: M
authors.lastname: Jia
authors.forename: Yun
authors.initials: Y
authors.lastname: Sun
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.forename: Feifei
authors.initials: F
authors.lastname: Wu
authors.forename: Hanfeng
authors.initials: H
authors.lastname: Zhang
authors.forename: Boya
authors.initials: B
authors.lastname: Wang
authors.forename: Michael K
authors.initials: MK
authors.lastname: Deyholos
authors.forename: Yuan-Qing
authors.initials: YQ
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D001203:Ascomycota / Q000502:physiology; D029688:Brassica napus / Q000201:enzymology* / Q000235:genetics* / Q000382:microbiology / Q000502:physiology; D003001:Cloning, Molecular; D042541:Intracellular Space / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013312:Stress, Physiological / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D014157:Transcription Factors; D020928:Mitogen-Activated Protein Kinases