, Volume 226, Issue 1, pp 73–85 | Cite as

Characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice

  • Yuemin Huang
  • Benze Xiao
  • Lizhong Xiong
Original Article


A full-length cDNA gene, designated Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1), was characterized in rice. The predicted protein of OCPI1 shows very high sequence identity to reported chymotrypsin inhibitors from various plant species. Northern-blot analysis showed that the expression of OCPI1 was strongly induced by dehydration stresses and abscisic acid (ABA). The expression of beta-glucuronidase (GUS) reporter gene under the control of OCPI1 promoter transformed into rice was strongly induced by drought and salt stresses. Interestingly, strong dehydration stress-induced GUS activity was also detected in the transgenic rice containing the reverse sequence of OCPI1 promoter fused to GUS gene, suggesting of a bidirectional transcriptional activity in the OCPI1 promoter. OCPI1 gene was over-expressed in japonica cv. Zhonghua 11 and transgenic plants containing single copy of transgene were tested for drought resistance at reproductive stage. The positive transgenic plants (OCPI1 was over-expressed) had significantly higher grain yield and seed setting rate than the wild type and the negative transgenic control (no over-expression of the transgene) under the severe drought stress conditions, whereas the potential yield of transgenic plants under normal growth conditions was not affected. Chymotrypsin-inhibitor activity assay showed that the crude protein of the positive transgenic plants had stronger inhibitory activity than the negative control. Transgenic plants had less decrease of total proteins than the wild type under drought stress. Taken together, these data indicate that OCPI1 might potentially be useful in the genetic improvement of drought resistance in rice.


Abiotic stress Drought resistance Promoter Proteinase inhibitor Oryza 



Abscisic acid




Hygromycin phosphotransferase


Proteinase inhibitor



This research was supported by grants partially from the National Basic Research Program of China, the National Natural Science Foundation of China, Commission of the European Communities (Contract No. INCO-015468) and the Rockefeller Foundation (2004FS070).


  1. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78PubMedCrossRefGoogle Scholar
  2. Barr HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficit in leaves. Aust J Biol Sci 15:413–428Google Scholar
  3. Botella MA, Xu Y, Prabha TN, Zhao Y, Narasimhan ML, Wilson KA, Nielsen SS, Bressan RA, Hasegawa PM (1996) Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate. Plant Physiol 112:1201–1210PubMedCrossRefGoogle Scholar
  4. Busk PK, Pages M (1998) Regulation of abscisic acid-induced transcription. Plant Mol Biol 37:425–435PubMedCrossRefGoogle Scholar
  5. Callis J (1995) Regulation of protein degradation. Plant Cell 7:845–857PubMedCrossRefGoogle Scholar
  6. Chen GH, Huang LT, Yap MN, Lee RH, Huang YJ, Cheng MC, Chen SC (2002) Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato. Plant Cell Physiol 43:984–991PubMedCrossRefGoogle Scholar
  7. Choi D, Park JA, Seo YS, Chun YJ, Kim WT (2000) Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L. Biochim Biophys Acta 1492:211–215PubMedGoogle Scholar
  8. Chu ZH, Peng KM, Zhang LD, Zhou B, Wei JaW SP (2003) Construction and characterization of a normalized whole-life-cycle cDNA library of rice. Chin Sci Bull 48:229–235CrossRefGoogle Scholar
  9. Clark AM, Jacobsen KR, Bostwick DE, Dannenhoffer JM, Skaggs MI, Thompson GA (1997) Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima. Plant J 12:49–61PubMedCrossRefGoogle Scholar
  10. Diop NN, Kidric M, Repellin A, Gareil M, d’Arcy-Lameta A, Pham Thi AT, Zuily-Fodil Y (2004) A multicystatin is induced by drought-stress in cowpea (Vigna unguiculata (L.) Walp.) leaves. FEBS Lett 577:545–550PubMedCrossRefGoogle Scholar
  11. Downing WL, Mauxion F, Fauvarque MO, Reviron MP, de Vienne D, Vartanian N, Giraudat J (1992) A Brassica napus transcript encoding a protein related to the Kunitz protease inhibitor family accumulates upon water stress in leaves, not in seeds. Plant J 2:685–693PubMedCrossRefGoogle Scholar
  12. Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot 51 Spec No:487–496Google Scholar
  13. El Maarouf H, Zuily-Fodil Y, Gareil M, d’Arcy-Lameta A, Pham-Thi AT (1999) Enzymatic activity and gene expression under water stress of phospholipase D in two cultivars of Vigna unguiculata L. Walp. differing in drought tolerance. Plant Mol Biol 39:1257–1265PubMedCrossRefGoogle Scholar
  14. Gaddour K, Vicente-Carbajosa J, Lara P, Isabel-Lamoneda I, Diaz I, Carbonero P (2001) A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli. Plant Mol Biol 45:599–608PubMedCrossRefGoogle Scholar
  15. Gosti F, Bertauche N, Vartanian N, Giraudat J (1995) Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana. Mol Gen Genet 246:10–18PubMedCrossRefGoogle Scholar
  16. Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71:83–112PubMedGoogle Scholar
  17. Habu Y, Fukushima H, Sakata Y, Abe H, Funada R (1996) A gene encoding a major Kunitz proteinase inhibitor of storage organs of winged bean is also expressed in the phloem of stems. Plant Mol Biol 32:1209–1213PubMedCrossRefGoogle Scholar
  18. Haq SK, Atif SM, Khan RH (2004) Protein proteinase inhibitor genes in combat against insects, pests, and pathogens: natural and engineered phytoprotection. Arch Biochem Biophys 431:145–159PubMedCrossRefGoogle Scholar
  19. Harrak H, Azelmat S, Baker EN, Tabaeizadeh Z (2001) Isolation and characterization of a gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum). Genome 44:368–374PubMedCrossRefGoogle Scholar
  20. Hendriks T, Vreugdenhil D, Stiekema WJ (1991) Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol Biol 17:385–394PubMedCrossRefGoogle Scholar
  21. Hibbetts K, Hines B, Williams D (1999) An overview of proteinase inhibitors. J Vet Intern Med 13:302–308PubMedCrossRefGoogle Scholar
  22. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282PubMedCrossRefGoogle Scholar
  23. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  24. 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
  25. Huang YJ, To KY, Yap MN, Chiang WJ, Suen DF, Chen SC (2001) Cloning and characterization of leaf senescence up-regulated genes in sweet potato. Physiol Plant 113:384–391PubMedCrossRefGoogle Scholar
  26. Huffaker RC (1990) Proteolytic activity during senescence of plants. New Phytol 116:199–231PubMedCrossRefGoogle Scholar
  27. Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403PubMedCrossRefGoogle Scholar
  28. IRRI (2004) World Rice Statistics. International Rice Research InstituteGoogle Scholar
  29. Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153PubMedCrossRefGoogle Scholar
  30. Jones ML, Larsen PB, Woodson WR (1995) Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol Biol 28:505–512PubMedCrossRefGoogle Scholar
  31. Kang SG, Choi JH, Suh SG (2002) A leaf-specific 27 kDa protein of potato Kunitz-type proteinase inhibitor is induced in response to abscisic acid, ethylene, methyl jasmonate, and water deficit. Mol Cells 13:144–147PubMedGoogle Scholar
  32. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291PubMedCrossRefGoogle Scholar
  33. Kleber-Janke T, Krupinska K (1997) Isolation of cDNA clones for genes showing enhanced expression in barley leaves during dark-induced senescence as well as during senescence under field conditions. Planta 203:332–340PubMedCrossRefGoogle Scholar
  34. Leung D, Abbenante G, Fairlie DP (2000) Protease inhibitors: current status and future prospects. J Med Chem 43:305–341PubMedCrossRefGoogle Scholar
  35. Lilley JM, Ludlow MM, McCouch SR, O’Toole JC (1996) Locating QTLs for osmotic adjustment and dehydration tolerance in rice. J Exp Bot 47:1427–1436CrossRefGoogle Scholar
  36. Lin YJ, Zhang Q (2005) Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23:540–547PubMedCrossRefGoogle Scholar
  37. Lopez F, Vansuyt G, Derancourt J, Fourcroy P, Casse-Delbart F (1994) Identification by 2D-page analysis of salt-stress induced proteins in radish (Raphanus sativus). Cell Mol Biol (Noisy-le-grand) 40:85–90Google Scholar
  38. Mosolov VV, Valueva TA (2005) Proteinase inhibitors and their function in plants: a review. Prikl Biokhim Mikrobiol 41:261–282PubMedGoogle Scholar
  39. Pena-Cortes H, Willmitzer L, Sanchez-Serrano JJ (1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell 3:963–972PubMedCrossRefGoogle Scholar
  40. Pernas M, Sanchez-Monge R, Salcedo G (2000) Biotic and abiotic stress can induce cystatin expression in chestnut. FEBS Lett 467:206–210PubMedCrossRefGoogle Scholar
  41. Price AH, Steele KAB, Moore J, Barraclough PP, Clark LJ (2000) A combined RFLP and AFLP linkage map of upland rice (Oryza sativa L.) lised to identify QTLs for root-penetration ability. Theor Appl Genet 100:49–56CrossRefGoogle Scholar
  42. Rakwal R, Kumar Agrawal G, Jwa NS (2001) Characterization of a rice (Oryza sativa L.) Bowman-Birk proteinase inhibitor: tightly light regulated induction in response to cut, jasmonic acid, ethylene and protein phosphatase 2A inhibitors. Gene 263:189–198PubMedCrossRefGoogle Scholar
  43. Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. Bioessays 10:20–24PubMedCrossRefGoogle Scholar
  44. Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K (2000) Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J 23:319–327PubMedCrossRefGoogle Scholar
  45. Satoh H, Uchida A, Nakayama K, Okada M (2001) Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein. Plant Cell Physiol 42:906–911PubMedCrossRefGoogle Scholar
  46. Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water-stress response. Plant Physiol 115:327–334PubMedCrossRefGoogle Scholar
  47. Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417PubMedCrossRefGoogle Scholar
  48. Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J 33:259–270PubMedCrossRefGoogle Scholar
  49. Sin SF, Yeung EC, Chye ML (2006) Downregulation of Solanum americanum genes encoding proteinase inhibitor II causes defective seed development. Plant J 45:58–70PubMedCrossRefGoogle Scholar
  50. Sin SF, Chye ML (2004) Expression of proteinase inhibitor II proteins during floral development in Solanum americanum. Planta 219:1010–1022PubMedCrossRefGoogle Scholar
  51. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–444PubMedCrossRefGoogle Scholar
  52. Su J, Shen Q, David Ho TH, Wu R (1998) Dehydration-stress-regulated transgene expression in stably transformed rice plants. Plant Physiol 117:913–922PubMedCrossRefGoogle Scholar
  53. Tran LS, Nakashima K, Sakuma Y, Simpson SD, Fujita Y, Maruyama K, Fujita M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell 16:2481–2498PubMedCrossRefGoogle Scholar
  54. Tripathy JN, Zhang J, Robin S, Nguyen HT (2000) QTLs for cell-membrane stability mapped in rice (Oryza sativa L.) under drought stress. Theor Appl Genet 100:1197–1202CrossRefGoogle Scholar
  55. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5:1529–1539PubMedCrossRefGoogle Scholar
  56. Vierstra RD (1996) Proteolysis in plants: mechanisms and functions. Plant Mol Biol 32:275–302PubMedCrossRefGoogle Scholar
  57. 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
  58. Xu D, Duan X, Wang B, Hong B, Ho T, Wu R (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 110:249–257PubMedGoogle Scholar
  59. Xu ZF, Teng WL, Chye ML (2004) Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a. Planta 218:623–629PubMedCrossRefGoogle Scholar
  60. 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
  61. Zagdanska B, Wisniewski K (1996) Endoproteinase activities in wheat leaves upon water deficit. Acta Biochim Pol 43:515–519PubMedGoogle Scholar
  62. Zhang J, Zheng HG, Aarti A, Pantuwan G, Nguyen TT, Tripathy JN, Sarial AK, Robin S, Babu RC, Nguyen BD, Sarkarung S, Blum A, Nguyen HT (2001) Locating genomic regions associated with components of drought resistance in rice: comparative mapping within and across species. Theor Appl Genet 103:19–29CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  1. 1.National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina

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