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Root-specific expression of a western white pine PR10 gene is mediated by different promoter regions in transgenic tobacco⋆

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Abstract

We report here the isolation and characterization of a novel PR10 gene, PmPR10-1.14, from western white pine (Pinus monticola Dougl. ex. D. Don). The PmPR10-1.14 gene encodes a polypeptide exhibiting high similarity with other members of the PR10 family and corresponds to one of six isoforms immunodetected in the roots of western white pine. Northern blot and western immunoblot analyses showed that expression of the PR10 gene family, including PmPR10-1.14, was detected in vegetative tissues constitutively, but not in developing reproductive organs. RT-PCR with gene-specific primers showed that the transcript of PmPR10-1.14 gene was found only in lateral roots and needles during growth. To study PR10 gene regulation at the cellular level, PmPR10-1.14 promoter was fused to the β-glucuronidase (GUS) report gene, and analyzed for transient and stable gene expression. The transient expression assays in agroinfiltrated tobacco leaves indicated that the core promoter of PmPR10-1.14 gene resided in the sequence from −101 to +69 relative to the first nucleotide of PR10 cDNA. Furthermore, the promoter region from −311 to −101 acted as an enhancer, and the region from −506 to −311 as a silencer. Fluorometric GUS assays of transgenic tobacco plants demonstrated that the longest promoter of 1675 bp directed GUS expression constitutively at high levels in the roots of mature plants, but expression levels were too low to be detectable in other organs in histochemical assays. Histochemical localization analysis showed that PmPR10-1.14 promoter directed a tissue-specific expression exclusively during the initiation and development of the lateral roots. The distal 5′ deletion of the promoter to −311 did not decrease the expression level significantly in the roots, suggesting that the cis-regulatory elements necessary for a high level of gene expression reside in the proximal fragment from −311 to +69. As one striking feature, PmPR10-1.14 promoter contains two copies of direct repeated sequences as long as 281 bp at its distal 5′ region. Deletion of one copy (−1326 to −1045) or both copies (−1675 to −1045) of the repeated sequences increased gene expression significantly in leaves and stems, which was regulated developmentally. Further deletion to −820 erased the increased gene expression in leaves and stems. These experiments revealed that the root-specific expression of PmPR10-1.14 gene is mediated by different promoter regions with both negative and positive regulatory mechanisms in transgenic tobacco plants.

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References

  • Bantignies, B., Séguin, J., Muzac, I., Dédaldéchamp, F., Gulick, P. and Ibrahim, R. 2000. Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots. Plant Mol. Biol. 42: 871–881.

    Google Scholar 

  • Barratt, D.H.P. and Clark, J.A. 1991. Proteins arising during the late stages of embryogenesis in Pisum sativum L. Planta 184: 14–23.

    Google Scholar 

  • Baumann, K., Paolis, A.D.P. and Gualberti, G. 1999. The DNA binding site of the Dof protein NtBBF1 is essential for tissue-specific and auxin-regulated expression of the rolB oncogene in plants. Plant Cell 11: 323–333.

    Google Scholar 

  • Benfey, P.N., Ren, L. and Chua, N.H. 1990. Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. EMBO J. 9: 1677–1684.

    Google Scholar 

  • Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O. and Breitenbach, M. 1989. The gene coding for the major birch pollen allergen Betv 1 is highly homologous to a pea disease resistance response gene. EMBO J. 8: 1935–1938.

    Google Scholar 

  • Breiteneder, H., Hoffmann-Sommergruber, K., O'Riordain, G., Susani, M., Ahorn, H., Ebner, C., Kraft, D. and Scheiner, O. 1995. Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens. Eur. J. Biochem. 233: 484–489.

    Google Scholar 

  • Bufe, A., Spangfort, M.D., Kahlert, H., Schlaak, M. and Becker, W.M. 1996. The major birch pollen allergen, bet v 1, shows ribonuclease activity. Planta 199: 413–415.

    Google Scholar 

  • Bureau, T.E. and Wessler, S.R. 1994. Stowaway: a new family of inverted repeat elements associated with the genes of both monocotyledonous and dicotyledonous plants. Plant Cell 6: 907–916.

    Google Scholar 

  • Constabel, C.P. and Brisson, N. 1995. Stigma-and vascular-specific expression of the PR10a gene of potato: a novel pattern of expression of a pathogenesis-related gene. Mol. Plant-Microbe Interact. 8: 104–113.

    Google Scholar 

  • Crowell, D.N., Maliyakal, E.J., Russell, D. and Amasino, R.M. 1992. Characterization of a stress-induced, developmentally regulated gene family from soybean. Plant Mol. Biol. 18: 459–466.

    Google Scholar 

  • Dubos, C. and Plomion, C. 2001. Drought differentially affects expression of a PR-10 protein in needles of the maritime pine (Pinus pinaster Ait.) seedling. J. Exp. Bot. 52: 1143–1144.

    Google Scholar 

  • Ekramoddoullah, A.K.M. and Davidson, J.J. 1995. A method for the determination of conifer foliage protein extracted using sodium dodecyl sulfate and mercaptoethanol. Phytochem. Anal. 6: 20–24.

    Google Scholar 

  • Ekramoddoullah, A.K.M. and Hunt, R.S. 1993. Changes in protein profile of susceptible and resistant sugar pine foliage infected with the white pine blister rust fungus Cronartium ribicola. Can. J. Plant Path. 15: 259–264.

    Google Scholar 

  • Ekramoddoullah, A.K.M. and Taylor, D. 1996. Seasonal variation of western white pine (Pinus monticola D. Don) foliage proteins. Plant Cell Physiol. 37: 189–199.

    Google Scholar 

  • Ekramoddoullah, A.K.M., Taylor, D. and Hawkins, B.J. 1995. Characterization of a fall protein of sugar pine and detection of its homologue associated with frost hardiness of western white pine needles. Can. J. For. Res. 25: 1137–1147.

    Google Scholar 

  • Ekramoddoullah, A.K.M., Davidson, J.J. and Taylor, D. 1998. A protein associated with frost hardiness of western white pine is up-regulated by infection in the white pine blister rust pathosystem. Can. J. For. Res. 28: 412–417.

    Google Scholar 

  • Ekramoddoullah, A.K.M., Yu, X., Sturrock, R., Zamani, A., and Taylor, D. 2000. Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglas-fir (Pseudotsuga menziesii) tissues. Physiol. Plant. 110: 240–247.

    Google Scholar 

  • Ellis, J.G., Llewellyn, D.J., Walker, J.C., Dennis, E.S. and Peacock, W.J. 1987. The ocs-element: a 16 base pair palindrome essential for activity of the octopine synthase enhancer. EMBO J. 6: 3203–3208.

    Google Scholar 

  • Feltkamp, D., Baumann, E., Schmalenbach, W., Masterson, R. and Rosahl, S. 1995. Expression of the mannopine synthase promoter in roots is dependent on the mas elements and correlates with high transcript leveles of mas-binding factor. Plant Sci. 109: 57–65.

    Google Scholar 

  • Fujimoto Y., Nagata, R., Fukasawa, H., Yano, K., Azuma, M., Iida, A., Sugimoyo, S., Shudo, K. and Hashimoto, Y. 1998. Purification and cDNA cloning of cytokinin-specific binding protein from mung bean (Vigna radiata). Eur. J. Biochem. 258: 794–802.

    Google Scholar 

  • Gajhede, M., Osmark, K., Poulsen, F.M., Ipsen, H., Larsen, J.N., Joost van Neerven, P.J., Schou, C., Løwenstein, H. and Spangfort, M.D. 1996. X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy. Nature Struct. Biol. 3: 1040–1045.

    Google Scholar 

  • Gonneau, M., Pagant, S., Brun, F. and Laloue, M. 2001. Photoaffinity labelling with the cytokinin agonist azido-CPPU of a 34 kDa peptide of the intracellular pathogenesis-related protein family in the moss Physcomitrella patens. Plant. Mol. Biol. 46: 539–548.

    Google Scholar 

  • Gubler, F., Raventos, D., Keys, M., Watts, R., Mundy, J. and Jacobsen, J.V. 1999. Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone. Plant J. 17: 1–9.

    Google Scholar 

  • Horsch, R.B., Fry, J.E., Hoffman, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. 1985. A simple and general method of transferring genes into plants. Science 227: 1229–1231.

    Google Scholar 

  • Huang, J.C., Chang, F.C. and Wang, C.S. 1997. Characterization of a lily tapetal transcript that shares sequence similarity with a class of intracellular pathogenesis-related (IPR) proteins. Plant Mol. Biol. 34: 681–686.

    Google Scholar 

  • Inaba, T., Nagano, Y., Sakakibara, T. and Sasaki, Y. 1999. Identification of a cis-regulatory element involved in phytochrome down-regulated expression of the pea small GTPase gene pra2. Plant Physiol. 120: 491–499.

    Google Scholar 

  • Jefferson, R.A. 1987. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405.

    Google Scholar 

  • Kobayashi, K., Fukuda, M., Igarashi, D. and Sunaoshi, M. 2001. Cytokinin-binding proteins from tobacco callus share homology with osmotin-like protein and an endochitinase. Plant Cell Physiol. 42: 148–157.

    Google Scholar 

  • Lam, E., Benfey, P.N., Gilmartin, P.M., Fang, R.X. and Chua, N.H. 1989. Site-specific mutations alter in vitro factor binding and change promoter expression pattern in transgenic plants. Proc. Natl. Acad. Sci. USA 86: 7890–7894.

    Google Scholar 

  • Lawton, M., Dean, S.M., Dron, M., Kooter, J.M., Kragh, K.M., Harrison, M.J., Yu, L., Tanguay, L., Dixon, R.A. and Lamb, C.J. 1991. Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1. Plant Mol. Biol. 16: 235–249.

    Google Scholar 

  • Linthorst, H.J.M. 1991. Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10: 123–150.

    Google Scholar 

  • Liu, J.-J., Goh, C.-J., Loh, C.-S., Liu, P. and Pua, E.-C. 1998. A method for isolation of total RNA from fruit tissues of banana. Plant Mol. Biol. Rep. 16: 87.

    Google Scholar 

  • Mascarenhas, D., Mettler, I.J., Pierce, D.A. and Lowe, H.W. 1990. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol. 15: 913–920.

    Google Scholar 

  • Matheus, Y.N., Ekramoddoullah, A.K.M. and Lee, S.P. 2000. Isolation and characterization of transcripts encoding putative intracellular pathogenesis-related protein from cold hardened white spruce tissues. Abstract S31–71, 6th International Congress of Plant Molecular Biology, 18–24 June 2000, Quebec, Canada.

  • Matton, D.P., and Brisson, N. 1989. Cloning, expression and sequence conservation of pathogenesis-related gene transcripts of potato. Mol. Plant-Microbe Interact. 2: 325–331.

    Google Scholar 

  • Martinez-Garcia, J.F., Moyano, E., Alcocer, M.J.C. and Martin, C. 1998. Two bZIP proteins from Antirrhium flowers preferentially bind to a hybrid C-box/G-box motif and help to define a new sub-family of bZIP transcription factors. Plant J. 13: 489–505.

    Google Scholar 

  • Moons, A., Prinsen, E., Bauw, G., and Van Montagu, M. 1997. Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots. Plant Cell 9: 2243–2259.

    Google Scholar 

  • Mylona, P., Moerman, M., Yang, W.-C., Gloudemans, T., van de Kerckhove, J., van Kammen, A., Bisseling, T. and Franssen, H.J. 1994. The root epidermis-specific pea gene RH2 is homologous to a pathogenesis-related gene. Plant Mol. Biol. 26: 39–50.

    Google Scholar 

  • Nap, J.-P., Keizer, P. and Jansen, R. 1993. First-generation transgenic plants and statistics. Plant Mol. Biol. Rep. 11: 156–164.

    Google Scholar 

  • Neave, H.R. and Worthington, P.L. 1988. Distribution-free Tests. Unwin Hyman, London.

    Google Scholar 

  • Quandt, K., Frech, K., Karas, H., Wingender, E. and Werner, T. 1995. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucl. Acids Res. 23: 4878–4884.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Plainview, NY.

    Google Scholar 

  • Sessa, G., Yang, X.Q., Raz, V., Eyal, Y. and Fluhr, R. 1995. Dark induction and subcellular localization of the pathogenesis-related PRB-1b protein. Plant Mol Biol. 28: 537–547.

    Google Scholar 

  • Sikorski, M.M., Biesiadka, J., Kasperska, A.E., Kopcinska, J., Lotocka, B., Golinowski, W. and Legocki, A. 1999. Expression of genes encoding PR10 class pathogenesis-related proteins is inhibited in yellow lupine root nodules. Plant Sci. 149: 125–137.

    Google Scholar 

  • Singh, K.H. 1998. Transcriptional regulation in plants: the importance of combinatorial control. Plant Physiol. 118: 1111–1120.

    Google Scholar 

  • Somssich, I.E., Schmelzer, E., Kawalleck, P. and Hahlbrock, K. 1988. Gene structure and its in situ transcript localization of pathogenesis-related protein 1 in parsley. Mol. Gen. Genet. 213: 93–98.

    Google Scholar 

  • Strömvik, M.V., Sundararaman, V.P. and Vodkin, L.O. 1999. A novel promoter from soybean that is active in a complex developmental pattern with and without its proximal 650 base pairs. Plant Mol. Biol. 41: 217–231.

    Google Scholar 

  • Swoboda, I., Hoffmann-Sommergruber, K., O.Ríordáin, G., Scheiner, O., Heberle-Bors, E. and Vincente, O. 1996. Bet v 1 proteins, the major birch pollen allergens and members of a family of conserved pathogenesis-related proteins, show ribonuclease activity in vitro. Physiol. Plant. 96: 433–438.

    Google Scholar 

  • Utriainen, M., Kokko, H., Auriola, S., Sarrazin, O. and Kärenlampi, S. 1998. PR-10 protein is induced by copper stress in roots and leaves of a Cu/Zn tolerant clone of birch, Betula pendula. Plant Cell Environ. 21: 821–828.

    Google Scholar 

  • Vanek-Krebitz, M., Hoffmann-Sommergruber, K., Laimer, D., Camara, M., Susani, M., Ebner, C., Kraft, D., Scheiner, O. and Breiteneder, H. 1995. Cloning and sequencing of Mald I, the major allergen from apple (Malus domestica), and its immunological relationship to Betv 1, the major birch pollen allergen. Biochem. Biophys. Res. Commun. 214: 538–551.

    Google Scholar 

  • van Loon, L.C., Pierpoint, W.S., Boller, T. and Conejero, V. 1994. Recommendation for naming plant pathogenesis-related proteins. Plant Mol. Biol. Rep. 12: 245–264.

    Google Scholar 

  • Vicente-Carbajosa, J., Moose, S.P., Parsons, R. and Schmidt, R.J. 1997. A maize zinc-finger protein binds the prolamin box in zein gene promoters and interacts with the basic leucine zipper transcriptional activator Opaque2. Pro. Natl. Acad. Sci. USA 94: 7685–7690.

    Google Scholar 

  • Walter, M.H., Liu, J.W., Grand, C., Lamb, C.J. and Hess, D. 1990. Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class of proteins including pollen allergens. Mol. Gen. Genet. 222: 353–360.

    Google Scholar 

  • Walter, M.H., Liu, J.W., Wünn, J. and Hess, D. 1996. Bean ribonuclease-like pathogenesis-related protein genes (Ypr10) display complex patterns of developmental, dark-induced and exogenous-stimulus-dependent expression. Eur. J. Biochem. 239: 281–293.

    Google Scholar 

  • Warner, S.A.J., Scott, R. and Draper, J. 1992. Characterization of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins. Plant Mol. Biol. 19: 555–561.

    Google Scholar 

  • Warner, S.A.J., Scott, R., and Draper, J. 1993. Isolation of an asparagus intracellular PR gene (AoPR1) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco. Plant J. 3: 191–201.

    Google Scholar 

  • Warner, S.A.J., Gill, A., and Draper, J. 1994. The developmental expression of the asparagus intracellular PR protein (AoPR1) gene correlates with sites of phenylpropanoid biosynthesis. Plant J. 6: 31–43.

    Google Scholar 

  • Wasylyk, B. 1988. Enhancers and transcription factors in the control of gene expression. Biochim. Biophys. Acta. 951: 17–35.

    Google Scholar 

  • Yanagisawa, S. 2000. Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J. 21: 281–288.

    Google Scholar 

  • Yanagisawa, S., and Schmidt, R.J. 1999. Diversity and similarity among recognition sequences of Dof transcription factors. Plant J. 17: 209–214.

    Google Scholar 

  • Yang, Y., Li, R. and Qi, M. 2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J. 22: 543–551.

    Google Scholar 

  • Yu, X., Ekramoddoullah, A.K.M., and Misra, S. 2000. Characterization of Pin m III cDNA in western white pine. Tree Physiol. 20: 663–671.

    Google Scholar 

  • Zhang, B., Chen, W., Foley, R.C., Büttner, M. and Singh, K.B. 1995. Interaction between distinct types of DNA binding proteins enhance binding to ocs element promoter sequences. Plant Cell 7: 2241–2252.

    Google Scholar 

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  1. Canadian Forest Service, Natural Resources Canada, Pacific Forestry Centre, 506 West Burnside Road, Victoria, BC, V8Z 1M5, Canada

    Jun-Jun Liu & Abul K.M. Ekramoddoullah

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Liu, JJ., Ekramoddoullah, A.K. Root-specific expression of a western white pine PR10 gene is mediated by different promoter regions in transgenic tobacco⋆ . Plant Mol Biol 52, 103–120 (2003). https://doi.org/10.1023/A:1023930326839

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