Plant Molecular Biology

, Volume 80, Issue 3, pp 241–253 | Cite as

OsWRKY30 is activated by MAP kinases to confer drought tolerance in rice

  • Huaishun Shen
  • Citao Liu
  • Yi Zhang
  • Xiuping Meng
  • Xin Zhou
  • Chengcai Chu
  • Xiping Wang


Both the WRKY transcription factor (TF) and MAP kinases have been shown to regulate gene expression in response to biotic and abiotic stresses in plants. Several reports have shown that WRKY TFs may function downstream of MAPK cascades. Here, we have shown that OsWRKY30 interacted with OsMPK3, OsMPK4, OsMPK7, OsMPK14, OsMPK20-4, and OsMPK20-5, and could be phosphorylated by OsMPK3, OsMPK7, and OsMPK14. Overexpression of OsWRKY30 in rice dramatically increased drought tolerance. Overexpression of OsWRKY30AA, in which all SP (serine residue followed by proline residue) sites were replaced by AP (A, alanine), resulted in no improvement in drought tolerance. In addition, the function of transcriptional activation of OsWRKY30 was impaired after SP was replaced by AP. These results proved that the phosphorylation of OsWRKY30 by MAPKs was crucial in order for OsWRKY30 to perform its biological function.


WRKY MAPK Drought tolerance Phosphorylation Transcriptional activation 



We greatly appreciate the laboratory conditions for in vitro phosphorylation assay, which was supplied by Dr. Yan Guo and the technical assistance by Dr. Huixin Lin (National Institute of Biological Sciences, Beijing, China). In addition, we would like to give our thanks to Prof. Haiyang Wang for the critical reading of and the valuable comments on this manuscript. This work was supported by grants from the Chinese National Natural Science Foundation (30770211), Agricultural Ministry of China (2008ZX08009-003), and the 863 High-tech Project from the Ministry of Science and Technology of China (2007AA10Z185).

Supplementary material

11103_2012_9941_MOESM1_ESM.doc (572 kb)
Supplementary material 1 (DOC 572 kb)


  1. Agalou A, Purwantomo S, Overnas E, Johannesson H, Zhu X, Estiati A, de Kam RJ, Engstrom P, Slamet-Loedin IH, Zhu Z, Wang M, Xiong L, Meijer AH, Ouwerkerk PB (2008) A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol 66:87–103PubMedCrossRefGoogle Scholar
  2. Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NHT, Zhu SJ, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Nielsen HB, Hirt H, Somssich I, Mattsson O, Mundy J (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589PubMedCrossRefGoogle Scholar
  3. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983PubMedCrossRefGoogle Scholar
  4. Chen SB, Tao LZ, Zeng LR, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein–protein interactions in rice. Mol Plant Pathol 7:417–427PubMedCrossRefGoogle Scholar
  5. Ciolkowski I, Wanke D, Birkenbihl RP, Somssich IE (2008) Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol 68:81–92PubMedCrossRefGoogle Scholar
  6. Cohen P (1997) The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol 7:353–361PubMedCrossRefGoogle Scholar
  7. Dong J, Chen C, Chen Z (2003) Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51:21–37PubMedCrossRefGoogle Scholar
  8. Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10:366–371PubMedCrossRefGoogle Scholar
  9. Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE (1999) Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors. EMBO J 18:4689–4699PubMedCrossRefGoogle Scholar
  10. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206PubMedCrossRefGoogle Scholar
  11. Fiil BK, Petersen K, Petersen M, Mundy J (2009) Gene regulation by MAP kinase cascades. Curr Opin Plant Biol 12:615–621PubMedCrossRefGoogle Scholar
  12. Hamel LP, Nicole MC, Sritubtim S, Morency MJ, Ellis M, Ehlting J, Beaudoin N, Barbazuk B, Klessig D, Lee J, Martin G, Mundy J, Ohashi Y, Scheel D, Sheen J, Xing T, Zhang S, Seguin A, Ellis BE (2006) Ancient signals: comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci 11:192–198PubMedCrossRefGoogle Scholar
  13. Hofgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877PubMedCrossRefGoogle Scholar
  14. Huang T, Duman JG (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48:339–350PubMedCrossRefGoogle Scholar
  15. Ishihama N, Yamada R, Yoshioka M, Katou S, Yoshioka H (2011) Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response. Plant Cell 23:1153–1170PubMedCrossRefGoogle Scholar
  16. Jonak C, Kiegerl S, Ligterink W, Barker PJ, Huskisson NS, Hirt H (1996) Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought. Proc Natl Acad Sci USA 93:11274–11279PubMedCrossRefGoogle Scholar
  17. Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195PubMedCrossRefGoogle Scholar
  18. Koo SC, Moon BC, Kim JK, Kim CY, Sung SJ, Kim MC, Cho MJ, Cheong YH (2009) OsBWMK1 mediates SA-dependent defense responses by activating the transcription factor OsWRKY33. Biochem Biophys Res Commun 387:365–370PubMedCrossRefGoogle Scholar
  19. Lee S, Jeon J-S, Jung K-H, An G (1999) Binary vectors for efficient transformation of rice. J Plant Biol 42:310–316CrossRefGoogle Scholar
  20. Ligterink W, Kroj T, zur Nieden U, Hirt H, Scheel D (1997) Receptor-mediated activation of a MAP kinase in pathogen defense of plants. Science 276:2054–2057Google Scholar
  21. Liu Y (2012) Roles of mitogen-activated protein kinase cascades in ABA signaling. Plant Cell Rep 31:1–12PubMedCrossRefGoogle Scholar
  22. Liu Y, Zhang S (2004) Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16:3386–3399PubMedCrossRefGoogle Scholar
  23. Mao G, Meng X, Liu Y, Zheng Z, Chen Z, Zhang S (2011) Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23:1639–1653PubMedCrossRefGoogle Scholar
  24. MAPKgroup (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7:301–308CrossRefGoogle Scholar
  25. Menke FLH, Kang HG, Chen ZX, Park JM, Kumar D, Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by the MAP kinase SIPK and mediates HR-like cell death in tobacco. Mol Plant Microbe Interact 18:1027–1034PubMedCrossRefGoogle Scholar
  26. Mishra NS, Tuteja R, Tuteja N (2006) Signaling through MAP kinase networks in plants. Arch Biochem Biophys 452:55–68PubMedCrossRefGoogle Scholar
  27. Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655PubMedCrossRefGoogle Scholar
  28. Pitzschke A, Djamei A, Teige M, Hirt H (2009) VIP1 response elements mediate mitogen-activated protein kinase 3-induced stress gene expression. Proc Natl Acad Sci USA 106:18414–18419PubMedCrossRefGoogle Scholar
  29. Qiu JL, Fiil BK, Petersen K, Nielsen HB, Botanga CJ, Thorgrimsen S, Palma K, Suarez-Rodriguez MC, Sandbech-Clausen S, Lichota J, Brodersen P, Grasser KD, Mattsson O, Glazebrook J, Mundy J, Petersen M (2008) Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. EMBO J 27:2214–2221PubMedCrossRefGoogle Scholar
  30. Rizhsky L, Liang H, Mittler R (2002) The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiol 130:1143–1151PubMedCrossRefGoogle Scholar
  31. Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev 16:1139–1149PubMedCrossRefGoogle Scholar
  32. Ross CA, Liu Y, Shen QXJ (2007) The WRKY gene family in rice (Oryza sativa). J Integr Plant Biol 49:827–842CrossRefGoogle Scholar
  33. Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:5690–5700PubMedGoogle Scholar
  34. Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258PubMedCrossRefGoogle Scholar
  35. Ryu HS, Han M, Lee SK, Cho JI, Ryoo N, Heu S, Lee YH, Bhoo SH, Wang GL, Hahn TR, Jeon JS (2006) A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response. Plant Cell Rep 25:836–847PubMedCrossRefGoogle Scholar
  36. Sanchez-Ballesta MT, Lluch Y, Gosalbes MJ, Zacarias L, Granell A, Lafuente MT (2003) A survey of genes differentially expressed during long-term heat-induced chilling tolerance in citrus fruit. Planta 218:65–70PubMedCrossRefGoogle Scholar
  37. Sarkar NK, Kim YK, Grover A (2009) Rice sHsp genes: genomic organization and expression profiling under stress and development. BMC Genom 10:393CrossRefGoogle Scholar
  38. Sheen J (2002) A transient expression assay using Arabidopsis mesophyll protoplasts.
  39. Sinha AK, Jaggi M, Raghuram B, Tuteja N (2011) Mitogen-activated protein kinase signaling in plants under abiotic stress. Plant Signal Behav 6:196–203PubMedCrossRefGoogle Scholar
  40. Takahashi R, Joshee N, Kitagawa Y (1994) Induction of chilling resistance by water stress, and cDNA sequence analysis and expression of water stress-regulated genes in rice. Plant Mol Biol 26:339–352PubMedCrossRefGoogle Scholar
  41. Tanoue T, Adachi M, Moriguchi T, Nishida E (2000) A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat Cell Biol 2:110–116PubMedCrossRefGoogle Scholar
  42. Tena G, Boudsocq M, Sheen J (2011) Protein kinase signaling networks in plant innate immunity. Curr Opin Plant Biol 14:519–529PubMedCrossRefGoogle Scholar
  43. Turck F, Zhou A, Somssich IE (2004) Stimulus-dependent, promoter-specific binding of transcription factor WRKY1 to its native promoter and the defense-related gene PcPR1-1 in Parsley. Plant Cell 16:2573–2585Google Scholar
  44. van Verk MC, Pappaioannou D, Neeleman L, Bol JF, Linthorst HJM (2008) A novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors. Plant Physiol 146:1983–1995PubMedCrossRefGoogle Scholar
  45. Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant Journal 40:428–438PubMedCrossRefGoogle Scholar
  46. Wu KL, Guo ZJ, Wang HH, Li J (2005) The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res 12:9–26PubMedCrossRefGoogle Scholar
  47. Wu X, Shiroto Y, Kishitani S, Ito Y, Toriyama K (2009) Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Rep 28:21–30PubMedCrossRefGoogle Scholar
  48. Xiong L, Yang Y (2003) Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell 15:745–759PubMedCrossRefGoogle Scholar
  49. Yang P, Chen C, Wang Z, Fan B, Chen Z (1999) A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter. Plant J 18:141–149CrossRefGoogle Scholar
  50. Zhang S, Klessig DF (1998) The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK. Proc Natl Acad Sci USA 95:7225–7230PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Huaishun Shen
    • 1
    • 2
  • Citao Liu
    • 2
    • 3
  • Yi Zhang
    • 2
  • Xiuping Meng
    • 2
  • Xin Zhou
    • 1
  • Chengcai Chu
    • 3
  • Xiping Wang
    • 2
  1. 1.Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research CenterChinese Academy of Fishery SciencesWuxiPeople’s Republic of China
  2. 2.National Engineering Research Center for Molecular Crop DesignBeijing Weiming Kaituo Agriculture Biotech Co., LtdBeijingPeople’s Republic of China
  3. 3.State Key Laboratory of Plant Genomics and Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations