Plant Molecular Biology

, Volume 77, Issue 6, pp 547–563 | Cite as

Characterization of an inositol 1,3,4-trisphosphate 5/6-kinase gene that is essential for drought and salt stress responses in rice

  • Hao Du
  • Linhong Liu
  • Lei You
  • Mei Yang
  • Yubing He
  • Xianghua Li
  • Lizhong Xiong


Drought and salt stresses are major limiting factors for crop production. To identify critical genes for stress resistance in rice (Oryza sativa L.), we screened T-DNA mutants and identified a drought- and salt-hypersensitive mutant dsm3. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative inositol 1,3,4-trisphosphate 5/6-kinase previously named OsITPK2 with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3 (OsITPK2) in rice resulted in drought- and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3 promoter-driven β-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3 is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3 to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2 is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice.


Abiotic stress Inositol phosphate Oryza IP3 Secondary signaling 



We thank Changyin Wu and (Huazhong Agricultural University, Wuhan, China) for providing the mutant dsm3, and Jan Xu and Rongjian Ye (Huazhong Agricultural University) for providing plasmid pM999-33 and DX2181, respectively. We also thank Yihua Zhou (Institute of Genetics and Development, Chinese Academy Science) for providing endoplasmic reticulum marker in subcellular localization assays. This work was supported by grants from the National Natural Science Foundation of China (30725021, 30921091, and 30830071), the National Program on the Development of Basic Research (2012CB114305), and the Project from the Ministry of Agriculture of China for Transgenic Research.

Supplementary material

11103_2011_9830_MOESM1_ESM.doc (7.8 mb)
Supplementary material 1 (DOC 8024 kb)


  1. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  2. Bennett M, Onnebo SM, Azevedo C, Saiardi A (2006) Inositol pyrophosphates: metabolism and signaling. Cell Mol Life Sci 63:552–564PubMedCrossRefGoogle Scholar
  3. Chittoor JM, Leach JE, White FF (1997) Differential induction of a peroxidase gene family during infection of rice by Xanthomonas oryzae pv. Oryzae. Mol Plant Microbe Interact 10:861–871Google Scholar
  4. DeWald DB, Torabinejad J, Jones CA, Shope JC, Cangelosi AR, Thompson JE, Prestwich GD, Hama H (2001) Rapid accumulation of phosphatidylinositol 4, 5-bisphosphate and inositol 1, 4, 5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis. Plant Physiol 126:759–769PubMedCrossRefGoogle Scholar
  5. Du H, Wang N, Cui F, Li X, Xiao J, Xiong L (2010) Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice. Plant Physiol 154:1304–1318PubMedCrossRefGoogle Scholar
  6. Dubois E, Scherens B, Vierendeels F, Ho MM, Messenguy F, Shears SB (2002) In Saccharomyces cerevisiae, the inositol polyphosphate kinase activity of Kcs1p is required for resistance to salt stress, cell wall integrity, and vacuolar morphogenesis. J Biol Chem 277:23755–23763PubMedCrossRefGoogle Scholar
  7. Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763PubMedCrossRefGoogle Scholar
  8. Field J, Wilson MP, Mai Z, Majerus PW, Samuelson J (2000) An Entamoeba histolytica inositol 1, 3, 4-trisphosphate 5/6-kinase has a novel 3-kinase activity. Mol Biochem Parasitol 108:119–123PubMedCrossRefGoogle Scholar
  9. Gardy JL, Spencer C, Wang K, Ester M, Tusnady GE, Simon I, Hua S, deFays K, Lambert C, Nakai K K, Brinkman FS (2003) PSORT-B: improving protein subcellular localization prediction for Gram-negative bacteria. Nucl Acids Res 31:3613–3617PubMedCrossRefGoogle Scholar
  10. Grover AK, Khan I (1992) Calcium pump isoforms: diversity, selectivity and plasticity. Review article. Cell Calcium 13:9–17PubMedCrossRefGoogle Scholar
  11. Gunesekera B, Torabinejad J, Robinson J, Gillaspy GE (2007) Inositol polyphosphate 5-phosphatases 1 and 2 are required for regulating seedling growth. Plant Physiol 143:1408–1417PubMedCrossRefGoogle Scholar
  12. Hill TD, Dean NM, Boynton AL (1988) Inositol 1, 3, 4, 5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells. Science 242:1176–1178PubMedCrossRefGoogle Scholar
  13. Ho MW, Yang X, Carew MA, Zhang T, Hua L, Kwon YU, Chung SK, Adelt S, Vogel G, Riley AM, Potter BV, Shears SB (2002) Regulation of Ins (3, 4, 5, 6)P(4) signaling by a reversible kinase/phosphatase. Curr Biol 12:477–482PubMedCrossRefGoogle Scholar
  14. Hong Z, Lakkineni K, Zhang Z, Verma DP (2000) Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136PubMedCrossRefGoogle Scholar
  15. Hou X, Xie K, Yao J, Qi Z, Xiong L (2009) A homolog of human ski-interacting protein in rice positively regulates cell viability and stress tolerance. Proc Natl Acad Sci USA 106:6410–6415PubMedCrossRefGoogle Scholar
  16. Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, Xiong L (2006) Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proc Natl Acad Sci USA 103:12987–12992PubMedCrossRefGoogle Scholar
  17. Hu H, You J, Fang Y, Zhu X, Qi Z, Xiong L (2008) Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol 67:169–181PubMedCrossRefGoogle Scholar
  18. Igarashi Y, Yoshiba Y, Sanada Y, Yamaguchi-Shinozaki K, Wada K, Shinozaki K (1997) Characterization of the gene for delta1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant Mol Biol 33:857–865PubMedCrossRefGoogle Scholar
  19. Josefsen L, Bohn L, Sorensen MB, Rasmussen SK (2007) Characterization of a multifunctional inositol phosphate kinase from rice and barley belonging to the ATP-grasp superfamily. Gene 397:114–125PubMedCrossRefGoogle Scholar
  20. Khodakovskaya M, Sword C, Wu Q, Perera IY, Boss WF, Brown CS, Winter Sederoff H (2010) Increasing inositol (1, 4, 5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato. Plant Biotech J 8:170–183CrossRefGoogle Scholar
  21. Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J (1988) The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332:462–464PubMedCrossRefGoogle Scholar
  22. Le Rudulier D, Strom AR, Dandekar AM, Smith LT, Valentine RC (1984) Molecular biology of osmoregulation. Science 224:1064–1068PubMedCrossRefGoogle Scholar
  23. Lin YJ, Zhang Q (2005) Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23:540–547PubMedCrossRefGoogle Scholar
  24. Liu J, Zhu JK (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114:591–596PubMedCrossRefGoogle Scholar
  25. Mignery GA, Johnston PA, Sudhof TC (1992) Mechanism of Ca2+ inhibition of inositol 1, 4, 5-trisphosphate (InsP3) binding to the cerebellar InsP3 receptor. J Biol Chem 267:7450–7455PubMedGoogle Scholar
  26. Mikoshiba K (1997) The InsP3 receptor and intracellular Ca2+ signaling. Curr Opin Neurobiol 7:339–345PubMedCrossRefGoogle Scholar
  27. Miller GJ, Wilson MP, Majerus PW, Hurley JH (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1, 3, 4-trisphosphate 5/6-kinase. Mol Cell 18:201–212PubMedCrossRefGoogle Scholar
  28. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410PubMedCrossRefGoogle Scholar
  29. Monnier V, Girardot F, Audin W, Tricoire H (2002) Control of oxidative stress resistance by IP3 kinase in Drosophila melanogaster. Free Radic Biol Med 33:1250–1259PubMedCrossRefGoogle Scholar
  30. Niu X, Chen Q, Wang X (2008) OsITL1 gene encoding an inositol 1, 3, 4-trisphosphate 5/6-kinase is a negative regulator of osmotic stress signaling. Biotechnol Lett 30:1687–1692PubMedCrossRefGoogle Scholar
  31. Perera IY, Hung CY, Brady S, Muday GK, Boss WF (2006) A universal role for inositol 1, 4, 5-trisphosphate-mediated signaling in plant gravitropism. Plant Physiol 140:746–760PubMedCrossRefGoogle Scholar
  32. Qin ZX, Chen QJ, Tong Z, Wang XC (2005) The Arabidopsis inositol 1, 3, 4-trisphosphate 5/6 kinase, AtItpk-1, is involved in plant photomorphogenesis under red light conditions, possibly via interaction with COP9 signalosome. Plant Physiol Biochem 43:947–954PubMedCrossRefGoogle Scholar
  33. Richardson A, Taylor CW (1993) Effects of Ca2+ chelators on purified inositol 1, 4, 5-trisphosphate (InsP3) receptors and InsP3-stimulated Ca2+ mobilization. J Biol Chem 268:11528–11533PubMedGoogle Scholar
  34. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574PubMedCrossRefGoogle Scholar
  35. Saiardi A, Cockcroft S (2008) Human ITPK1: a reversible inositol phosphate kinase/phosphatase that links receptor-dependent phospholipase C to Ca2+ -activated chloride channels. Sci Signal 1:pe5PubMedCrossRefGoogle Scholar
  36. Shen X, Liu H, Yuan B, Li X, Xu C, Wang S (2011) OsEDR1 negatively regulates rice bacterial resistance via activation of ethylene biosynthesis. Plant Cell Environ 34:179–191PubMedCrossRefGoogle Scholar
  37. Shi J, Wang H, Wu Y, Hazebroek J, Meeley RB, Ertl DS (2003) The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol 131:507–515PubMedCrossRefGoogle Scholar
  38. Stevenson JM, Perera IY, Heilmann II, Persson S, Boss WF (2000) Inositol signaling and plant growth. Trends Plant Sci 5:357PubMedCrossRefGoogle Scholar
  39. Sun Y, Wilson MP, Majerus PW (2002) Inositol 1, 3, 4-trisphosphate 5/6-kinase associates with the COP9 signalosome by binding to CSN1. J Biol Chem 277:45759–45764PubMedCrossRefGoogle Scholar
  40. Suzuki M, Tanaka K, Kuwano M, Yoshida KT (2007) Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway. Gene 405:55–64PubMedCrossRefGoogle Scholar
  41. Sweetman D, Stavridou I, Johnson S, Green P, Caddick SE, Brearley CA (2007) Arabidopsis thaliana inositol 1, 3, 4-trisphosphate 5/6-kinase 4 (AtITPK4) is an outlier to a family of ATP-grasp fold proteins from Arabidopsis. FEBS Lett 581:4165–4171PubMedCrossRefGoogle Scholar
  42. Takazawa K, Perret J, Dumont JE, Erneux C (1991) Molecular cloning and expression of a new putative inositol 1, 4, 5-trisphosphate 3-kinase isoenzyme. Biochem J 278(Pt 3):883–886PubMedGoogle Scholar
  43. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  44. Tuteja N, Mahajan S (2007) Calcium signaling network in plants: an overview. Plant Signal Behav 2:79–85PubMedCrossRefGoogle Scholar
  45. Wang L, Xie W, Chen Y, Tang W, Yang J, Ye R, Liu L, Lin Y, Xu C, Xiao J, Zhang Q (2010) A dynamic gene expression atlas covering the entire life cycle of rice. Plant J 61:752–766PubMedCrossRefGoogle Scholar
  46. Warthmann N, Chen H, Ossowski S, Weigel D, Herve P (2008) Highly specific gene silencing by artificial miRNAs in rice. PLoS One 3:e1829PubMedCrossRefGoogle Scholar
  47. Wilson MP, Majerus PW (1997) Characterization of a cDNA encoding Arabidopsis thaliana inositol 1, 3, 4-trisphosphate 5/6-kinase. Biochem Biophys Res Commun 232:678–681PubMedCrossRefGoogle Scholar
  48. Wilson MP, Sun Y, Cao L, Majerus PW (2001) Inositol 1, 3, 4-trisphosphate 5/6-kinase is a protein kinase that phosphorylates the transcription factors c-Jun and ATF-2. J Biol Chem 276:40998–41004PubMedCrossRefGoogle Scholar
  49. Wu C, Li X, Yuan W, Chen G, Kilian A, Li J, Xu C, Li X, Zhou DX, Wang S, Zhang Q (2003) Development of enhancer trap lines for functional analysis of the rice genome. Plant J 35:418–427PubMedCrossRefGoogle Scholar
  50. Xiang Y, Huang Y, Xiong L (2007) Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol 144:1416–1428PubMedCrossRefGoogle Scholar
  51. Xiang Y, Tang N, Du H, Ye H, Xiong L (2008) Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice. Plant Physiol 148:1938–1952PubMedCrossRefGoogle Scholar
  52. Xiao B, Huang Y, Tang N, Xiong L (2007) Over-expression of a LEA gene in rice improves drought resistance under the field conditions. Theor Appl Genet 115:35–46PubMedCrossRefGoogle Scholar
  53. Xiong L, Lee B, Ishitani M, Lee H, Zhang C, Zhu JK (2001) FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev 15:1971–1984PubMedCrossRefGoogle Scholar
  54. Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14(Suppl):S165–S183PubMedGoogle Scholar
  55. Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222PubMedCrossRefGoogle Scholar
  56. Yang X, Rudolf M, Carew MA, Yoshida M, Nerreter V, Riley AM, Chung SK, Bruzik KS, Potter BV, Schultz C, Shears SB (1999) Inositol 1, 3, 4-trisphosphate acts in vivo as a specific regulator of cellular signaling by inositol 3, 4, 5, 6-tetrakisphosphate. J Biol Chem 274:18973–18980PubMedCrossRefGoogle Scholar
  57. Yang L, Tang R, Zhu J, Liu H, Mueller-Roeber B, Xia H, Zhang H (2008) Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2beta, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana. Plant Mol Biol 66:329–343PubMedCrossRefGoogle Scholar
  58. Ye H, Du H, Tang N, Li X, Xiong L (2009) Identification and expression profiling analysis of TIFY family genes involved in stress and phytohormone responses in rice. Plant Mol Biol 71:291–305PubMedCrossRefGoogle Scholar
  59. Yue B, Xue W, Xiong L, Yu X, Luo L, Cui K, Jin D, Xing Y, Zhang Q (2006) Genetic basis of drought resistance at reproductive stage in rice: separation of drought tolerance from drought avoidance. Genetics 172:1213–1228PubMedCrossRefGoogle Scholar
  60. Zhang J, Li C, Wu C, Xiong L, Chen G, Zhang Q, Wang S (2006) RMD: a rice mutant database for functional analysis of the rice genome. Nucl Acids Res 34:D745–D748PubMedCrossRefGoogle Scholar
  61. Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273PubMedCrossRefGoogle Scholar
  62. Ziolkowski N, Grover AK (2010) Functional linkage as a direction for studies in oxidative stress: alpha-adrenergic receptors. Can J Physiol Pharmacol 88:220–232PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Hao Du
    • 1
  • Linhong Liu
    • 1
  • Lei You
    • 1
  • Mei Yang
    • 1
  • Yubing He
    • 1
  • Xianghua Li
    • 1
  • Lizhong Xiong
    • 1
  1. 1.National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan)Huazhong Agricultural UniversityWuhanChina

Personalised recommendations