Abstract
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.
Similar content being viewed by others
Abbreviations
- ABA:
-
Abscisic acid
- GUS:
-
Beta-glucuronidase
- Hpt:
-
Hygromycin phosphotransferase
- PI:
-
Proteinase inhibitor
References
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 15:63–78
Barr HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficit in leaves. Aust J Biol Sci 15:413–428
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 112:1201–1210
Busk PK, Pages M (1998) Regulation of abscisic acid-induced transcription. Plant Mol Biol 37:425–435
Callis J (1995) Regulation of protein degradation. Plant Cell 7:845–857
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 43:984–991
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 1492:211–215
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 48:229–235
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 12:49–61
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 577:545–550
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 2:685–693
Dunwell JM (2000) Transgenic approaches to crop improvement. J Exp Bot 51 Spec No:487–496
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 39:1257–1265
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 45:599–608
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 246:10–18
Guo Y, Gan S (2005) Leaf senescence: signals, execution, and regulation. Curr Top Dev Biol 71:83–112
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 32:1209–1213
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 431:145–159
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 44:368–374
Hendriks T, Vreugdenhil D, Stiekema WJ (1991) Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development. Plant Mol Biol 17:385–394
Hibbetts K, Hines B, Williams D (1999) An overview of proteinase inhibitors. J Vet Intern Med 13:302–308
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 6:271–282
Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300
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 103:12987–12992
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 113:384–391
Huffaker RC (1990) Proteolytic activity during senescence of plants. New Phytol 116:199–231
Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403
IRRI (2004) World Rice Statistics. International Rice Research Institute
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 47:141–153
Jones ML, Larsen PB, Woodson WR (1995) Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol Biol 28:505–512
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 13:144–147
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 17:287–291
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 203:332–340
Leung D, Abbenante G, Fairlie DP (2000) Protease inhibitors: current status and future prospects. J Med Chem 43:305–341
Lilley JM, Ludlow MM, McCouch SR, O’Toole JC (1996) Locating QTLs for osmotic adjustment and dehydration tolerance in rice. J Exp Bot 47:1427–1436
Lin YJ, Zhang Q (2005) Optimising the tissue culture conditions for high efficiency transformation of indica rice. Plant Cell Rep 23:540–547
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) 40:85–90
Mosolov VV, Valueva TA (2005) Proteinase inhibitors and their function in plants: a review. Prikl Biokhim Mikrobiol 41:261–282
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 3:963–972
Pernas M, Sanchez-Monge R, Salcedo G (2000) Biotic and abiotic stress can induce cystatin expression in chestnut. FEBS Lett 467:206–210
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 100:49–56
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 263:189–198
Ryan CA (1989) Proteinase inhibitor gene families: strategies for transformation to improve plant defenses against herbivores. Bioessays 10:20–24
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 23:319–327
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 42:906–911
Shinozaki K, Yamaguchi-Shinozaki K (1997) Gene expression and signal transduction in water-stress response. Plant Physiol 115:327–334
Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417
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 33:259–270
Sin SF, Yeung EC, Chye ML (2006) Downregulation of Solanum americanum genes encoding proteinase inhibitor II causes defective seed development. Plant J 45:58–70
Sin SF, Chye ML (2004) Expression of proteinase inhibitor II proteins during floral development in Solanum americanum. Planta 219:1010–1022
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 11:431–444
Su J, Shen Q, David Ho TH, Wu R (1998) Dehydration-stress-regulated transgene expression in stably transformed rice plants. Plant Physiol 117:913–922
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 16:2481–2498
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 100:1197–1202
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 5:1529–1539
Vierstra RD (1996) Proteolysis in plants: mechanisms and functions. Plant Mol Biol 32:275–302
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 15:745–759
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 110:249–257
Xu ZF, Teng WL, Chye ML (2004) Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a. Planta 218:623–629
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 172:1213–1228
Zagdanska B, Wisniewski K (1996) Endoproteinase activities in wheat leaves upon water deficit. Acta Biochim Pol 43:515–519
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 103:19–29
Acknowledgments
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).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Huang, Y., Xiao, B. & Xiong, L. Characterization of a stress responsive proteinase inhibitor gene with positive effect in improving drought resistance in rice. Planta 226, 73–85 (2007). https://doi.org/10.1007/s00425-006-0469-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-006-0469-8