Plant Molecular Biology

, Volume 44, Issue 5, pp 649–657 | Cite as

Circadian and senescence-enhanced expression of a tobacco cysteine protease gene

  • Tadamasa Ueda
  • Shigemi Seo
  • Yuko Ohashi
  • Junji Hashimoto


A cDNA clone encoding a cysteine protease was isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from degenerated primers based on the conserved sequences of plant cysteine protease genes. A putative protein encoded by the clone NTCP-23 had an amino acid sequence with significant similarities to those of plant senescence-associated cysteine proteases and mammalian cathepsin H. Northern blot analysis showed that NTCP-23 mRNA is expressed in all organs and the mRNA and protein expression is enhanced during natural senescence. We propose that NTCP-23 is responsible for amino acid remobilization especially in senescencing leaves. Furthermore, it was found that the mRNA expression follows a circadian rhythm and is reduced by continuous darkness, wounding and hypersensitive reaction (HR). NTCP-23 is the first cysteine protease whose mRNA expression has been shown to be temporarily reduced by wounding.

circadian rhythm cysteine protease (cysteine endopeptidases EC 3.4.22) senescence tobacco (Nicotiana tabacumwounding 


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  1. Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24: 1–15.Google Scholar
  2. Balandin, T., van der Does, C., Albert, J.M., Bol, J.F. and Linthorst, H.J.M. 1995. Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco. Plant Mol. Biol. 27: 1197–1204.PubMedGoogle Scholar
  3. Biswal, U.C., Biswal, B. 1984 Photocontrol of leaf senescence. Photochem. Photobiol. 39: 875–879.Google Scholar
  4. Cercos, M., Santamaria, S. and Carbonell, J. 1999. Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea. Plant Physiol. 119: 1341–1348.PubMedGoogle Scholar
  5. Cervantes, E., Rodriguez, A. and Nicolas, G. 1994. Ethylene regulates the expression of a cysteine protease gene during germination of chickpea (Cicer arietinum L.). Plant Mol. Biol. 25: 207–215.PubMedGoogle Scholar
  6. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299.PubMedGoogle Scholar
  7. Cimerman, N., Brguljan, P.M., Krasovec, M., Suskovic, S. and Kos, J. 1999. Circadian characteristics of cathepsins B, H, L, and stefins A and B, potential markers for disease, in nomal sera. Clin. Chim. Acta 282: 211–218.PubMedGoogle Scholar
  8. Drake, R., John, I., Farrell, A., Cooper, W., Schuch, W., and Grierson, D. 1996. Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence. Plant Mol. Biol. 30: 755–767.PubMedGoogle Scholar
  9. Forsthoefel, N.R., Cushman, M.A.F., Ostrem, J.A. and Cushman, J.C. 1998. Induction of a cysteine protease cDNA from Mesembryanthemum crystallinum leaves by environmental stress and plant growth regulators. Plant Sci. 136: 195–206.Google Scholar
  10. Fuchs, R. and Gassen, H.G. 1989 Nucleotide sequence of human preprocathepsin H, a lysosomal cysteine proteinase. Nucl. Acids Res. 17: 9471–9471.PubMedGoogle Scholar
  11. Garbarino, J.E., Oosumi, T. and Belknap, W.R. 1995. Isolation of a polyubiquitin promoter and its expression in transgenic potato plants. Plant Physiol. 109: 1371–1378.PubMedGoogle Scholar
  12. Giuliano, G., Hoffman, N.E., Ko, K., Scolnik, P.A. and Cashmore, A.R. 1988 A light-enhanced circadian clock controls transcription of several plant genes. EMBO J. 7: 3635–3642.PubMedGoogle Scholar
  13. Gonzales, R.G., Haxo, R.S., and Scheich, T. 1980. Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein-nucleic acid interaction. Biochemistry 19: 4299–4303.PubMedGoogle Scholar
  14. Granell, A., Harris, N., Pisabarro, A.G. and Carbonell, J. 1992. Temporal and spatial expression of a thiolprotease gene during pea ovary senescence and its regulation by gibberellin. Plant J. 2: 907–915.PubMedGoogle Scholar
  15. Griffiths, C.M., Hosken, S.E., Oliver, D., Chojecki, A.J.S. and Thomas, H. 1997. Sequencing, expression pattern and RFLP mapping of a senescence-enhanced cDNA from Zea mays with high homology to oryzain and aleurain. Plant Mol. Biol. 34: 815–821.PubMedGoogle Scholar
  16. Guerrero, C., de la Calle, M., Reid, M.S. and Valpuesta, V. 1998. Analysis of the expression of two thiol protease genes from daylily (Hemerocallis spp.) during flower senescence. Plant. Mol. Biol. 36: 565–571.PubMedGoogle Scholar
  17. Hensel, L.L., Grbic, V., Baumgarten, D.A. and Bleeker, A.B. 1993. Developmental and age-related process that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5: 553–564.CrossRefPubMedGoogle Scholar
  18. Holwerda, B.C. and Rogers, J.C. 1993. Structure, functional properties and vacuolar targeting of the barley thiol protease, aleurain. J. Exp. Bot. 44: 321–329.Google Scholar
  19. Ito, N., Tomizawa, K., Tanaka, K., Matsui, M., Kendrick, R.E., Sato, T. and Nakagawa, H. 1997. Characterization of 26S proteasome β-and β-type and ATPase subunits from spinach and their expression during early stages of seedling developmant. Plant Mol. Biol. 34: 307–316.PubMedGoogle Scholar
  20. Jones, M.L., Larsen, P.B. and Woodson, W.R. 1995. Ethyleneregulated expression of a carnation cysteine proteinase during flower petal senescence. Plant Mol. Biol. 28: 505–512.PubMedGoogle Scholar
  21. Karrer, K.M., Peiffer, S.L. and DiTomas, M.E. 1993. Two distinct gene subfamilies within the family of cysteine protease genes. Proc. Natl. Acad. Sci. USA 90: 3063–3067.PubMedGoogle Scholar
  22. Klever-Janke, T. and 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.PubMedGoogle Scholar
  23. Koizumi, M., Yamaguchi-Shinozaki, K., Tsuji, H., and Shinozaki, K. 1993. Structure and expression of two genes that encode 657 distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene 129: 175–182.Google Scholar
  24. Koltunow, A.M., Truettner, J., Cox, K.H., Wallroth, M. and Goldberg, R.B. 1990. Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2: 1201–1224.CrossRefPubMedGoogle Scholar
  25. Lidgett, A.J., Moran, M., Wong, K.A.L., Furze, J., Rhodes, M.J.C. and Hamill, J.D. 1995. Isolation and expression pattern of a cDNA encoding a cathepsin B-like protease from Nicotiana rustica. Plant Mol. Biol. 29: 379–384.PubMedGoogle Scholar
  26. Linthorst, H.J.M., van der Does, C., Brederode, F.T. and Bol, J.F. 1993. Circadian expression and induction by wounding of tobacco genes for cysteine proteinase. Plant Mol. Biol. 21: 685–694.PubMedGoogle Scholar
  27. Lohman, K.N., Gan, S., John, M.C. and Amasino, R.M. 1994. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant. 92: 322–328.Google Scholar
  28. Martino-Catt, S. and Ort, D.R. 1992. Low temperature interrupts circadian regulation of transcriptional activity in chilling-sensitive plants. Proc. Natl. Acad. Sci. USA 89: 3731–3735.PubMedGoogle Scholar
  29. Millar, A.J. and Kay, S.A. 1991, Circadian control of cab gene transcription and mRNA accumulation in Arabidopsis. Plant Cell 3: 541–550.CrossRefPubMedGoogle Scholar
  30. Morris, K., Thomas, H. and Rogers, L. 1996. Endopeptidases during the development and senescence of Lolium temulentum leaves. Phytochemistry 4: 377–384.Google Scholar
  31. Oh, S.A., Lee, S.Y., Chung, I.K., Lee, C.H. and Nam, H.G. 1996. A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence. Plant Mol. Biol. 30: 739–754.PubMedGoogle Scholar
  32. Ohtsubo, N., Mitsuhara, I., Koga, M., Seo, S. and Ohashi Y. 1999. Ethylene promotes the necrotic lesion formation and basic PR gene expression in TMV-infected tobacco. Plant Cell Physiol 40: 808–817.Google Scholar
  33. Park, J.H., Oh, S.A., Kim, Y.H., Woo, H.R. and Nam, H.G. 1998. Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in Arabidopsis. Plant Mol. Biol. 37: 445–454.PubMedGoogle Scholar
  34. Pichersky, E., Bernatzky, R., Tanksley, S.D., Breidenbach, R.B., Kausch, A.R. and Cashmore, A.R. 1985. Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato). Gene 40: 247–258.PubMedGoogle Scholar
  35. Rogers, J.C., Dean, D. and Heck, G.R. 1985. Areurain: a barley thiol protease closely related to mammalian cathepsin H. Proc. Natl. Acad. Sci. USA 82: 6512–6516.PubMedGoogle Scholar
  36. Schaffer, M.A. and Fischer, R.L. 1988. Analysis of mRNAs that accumulate in response to low temperature identifies a thiol protease gene in tomato. Plant Physiol. 87: 431–436.Google Scholar
  37. Schwartz, W.N. and Barrett, A.J. 1980. Human cathepsin H. Biochem. J. 191: 487–497.PubMedGoogle Scholar
  38. Solomon, M., Belenghi, B., Delledonne, M., Manachem, E. and Levine, A. 1999. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11: 431–443.PubMedGoogle Scholar
  39. Tournaire, C., Kushnir, S., Bauw, G., Inzé, D., Teyssendier de la Serve, B. and Renaudin J.P. 1996. A thiol protease and an anionic peroxidase are induced by lowering cytokinins during callus growth in Petunia. Plant Physiol. 111: 159–168.PubMedGoogle Scholar
  40. Valpuesta, V., Lange, N., Guerrero, C. and Reid, M. 1995. Up-regulation of a cysteine protease accompanies the ethyleneinsensitive senescence of daylily (Hemerocallis) flowers. Plant Mol. Biol. 28: 575–582.PubMedGoogle Scholar
  41. Vierstra, R.D. 1996. Proteolysis in plant: mechanisms and functions. Plant Mol. Biol. 32: 275–302.PubMedGoogle Scholar
  42. Watanabe, H., Abe, K., Emori, Y., Hosoyama, H. and Arai, S. 1991. Molecular cloning and gibberellin-induced expression of multiple cysteine proteinase of rice seeds (oryzains). J. Biol. Chem. 266: 16897–16902.PubMedGoogle Scholar
  43. Wittenbach, V.A. 1977. Induced senescence of intact wheat seedlings and its reversibility. Plant Physiol. 59: 1039–1042.Google Scholar
  44. Wittenbach, V.A. 1978. Breakdown of ribulose bisphophate carboxylase and change in proteolytic activity during dark-induced senescence of wheat seedlings. Plant Physiol. 62: 604–608.Google Scholar
  45. Xu, F.X. and Chye, M.L. 1999. Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J. 17: 321–327.PubMedGoogle Scholar
  46. Ye, Z.-H. and Varner, J.E. 1996. Induction of cysteine and serine protease during xylogenesis in Zinnia elegans. Plant Mol. Biol. 30: 1233–1246.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Tadamasa Ueda
    • 1
    • 2
  • Shigemi Seo
    • 1
    • 2
  • Yuko Ohashi
    • 1
    • 2
  • Junji Hashimoto
    • 1
    • 2
  1. 1.National Institute of Agrobiological ResourcesIbarakiJapan
  2. 2.CRESTJapan Science and Technology Corporation, ChiyodakuTokyoJapan

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