Skip to main content
Log in

A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

A barley cDNA clone encoding a cysteine proteinase inhibitor was characterized. The deduced amino acid sequence of this barley cystatin (Hv-CPI) contains the motif QXVXG conserved among members of the cystatin superfamily. The gene (Icy), located on chromosome 2, was expressed in embryos, developing endosperms, leaves and roots as assessed by northern blot analysis. Western blot analysis detected a slightly retarded band in leaves that was not present in roots or seeds. In these two organs a more precise location of Hv-CPI was done by immuno-histochemical analysis, with polyclonal antibodies raised against the recombinant CPI protein expressed in Escherichia coli. This protein efficiently inhibited papain (K i 2.0×10−8 M) and ficin (K i 2.2×10−8 M) and, to a lesser extent, chymopapain (K i 1.6×10−7 M) and was inactive against bromelain. The Icy mRNA expression in vegetative tissues increased in response to anaerobiosis, dark and cold shock (6 °C).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • Abe, M. and Arai, S. 1991. Some properties of a cysteine proteinase inhibitor from corn endosperm.Agric. Biol. Chem.55: 2417–1418.

    Google Scholar 

  • Abe, M., Emori, Y., Kondo, H., Suzuki, K. and Arai, S. 1987a. Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin).J. Biol. Chem.262: 16793–16797.

    Google Scholar 

  • Abe, K., Kondo, H., Arai, S. 1987b. Purification and characterization of a rice cysteine proteinase inhibitor.Agric. Biol. Chem. 51:2763–2768.

    Google Scholar 

  • Abe, M., Abe, K., Kuroda, M. and Arai, S. 1992. Corn kernel cysteine proteinase inhibitor as a novel cystatin superfamily member of plant origin: molecular cloning and expression studies.FEBS Lett. 209:932–937.

    Google Scholar 

  • Abe, M., Abe, K., Iwabuchi, K, Domoto, C. and Arai, S. 1994. Corn cystatin I expressed in Escherichia coli: investigation of its inhibitory profile and occurrence in corn kernels.J. Biochem. 116: 488–492.

    Google Scholar 

  • Arai, S., Watanabe, H., Kondo, H., Emori, Y. and Abe, K. 1991. Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members.J. Biochem. 109:294–298.

    Google Scholar 

  • Bolter, C.J. 1993.Methyl jasmonate induces papain inhibitor(s) in tomato leaves.Plant Physiol.103: 1347–1353.

    Google Scholar 

  • Botella, M.A., Xu, Y., Prabha, T.N., Zhao, Y., Narasimhan, M.L., Wilson, A., Nielsen, S.S., Bressan, R.A and Hasegawa, E.P. 1996.Differential expression of soybean cysteine proteinase inhibitor genes during development and in response to wounding and methyl jasmonate.Plant Physiol.112: 1201–1210.

    Google Scholar 

  • Brzin, J., Popovic, T., Ritonja, A., Puizdar, V. and Kidric, M. 1998. Related cystatin inhibitors from leaf and from seed of Phaseolus vulgaris L. Plant Sci. 138:17–26.

    Google Scholar 

  • Cejudo, F.J., Murphy, G., Chinoy, C. and Baulcombe, D.C. 1992. A gibberelin-regulated gene from wheat with sequence homology to cathepsin B of mammalian cells.Plant J.2: 937–948.

    Google Scholar 

  • Dopico, B., Lowe, A.L., Wilson, I.D., Merodio, C. and Grierson D. 1993.Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage. Plant Mol. Biol. 21:437–449.

    Google Scholar 

  • Gruden, K., Strukelj, B., Ravnikar, M., Poljsak-Prijatelj, M., Mavric, I., Brzin, J., Pungercar, J. and Kregar, I. 1997.Potato cysteine proteinase inhibitor gene family: molecular cloning, characterization and immunocytochemical localisation studies. Plant Mol. Biol.34:317–323.

    Google Scholar 

  • Guerin, J. and Carbonero, P. 1997. The spatial distribution of su-crose synthase isoenzymes in barley.Plant Physiol.114:55–62.

    Google Scholar 

  • Hirashiki, I., Ogata, F., Yoshida, N., Makisumi, S. and Ito, A. 1990. Purification and complex formation analysis of a cysteine proteinase inhibitor (cystatin) from seeds of Wisteria floribunda.J. Biochem. 108:604–608.

    Google Scholar 

  • Ievleva, E.V., Rudenskaya, Y.A., Zimacheva, A.V. and Mosolov, V.V. 1995.Cystatin from barley seeds.Biochemistry (Moscow) 60:1237–1240.

    Google Scholar 

  • Kimura, K., Ikeda, T., Fukumoto, D., Yamasaki, N. and Yonekura, M. 1995. Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill).Biosci. Biotechnol. Biochem. 59:2328–2329.

    Google Scholar 

  • Kondo, H., Abe, K., Nishimura, I., Watanabe, H., Emori, Y. and Arai, S. 1990. Two distinct cystatin in rice seeds with different specificities against cysteine proteinases.J. Biol. Chem. 265: 15832–15837.

    Google Scholar 

  • Kouzuma, Y., Kawano, K., Kimura, M., Yamasaki, N., Kadowaki, T. and Yamamoto, K. 1996.Purification, characterization and sequencing of two cysteine proteinase inhibitors, Sca and Scb, from sunflower (Helianthus annuus) seeds.J. Biochem.119: 1106–1113.

    Google Scholar 

  • Kuroda, M., Ishimoto, M., Suzuki, K., Kondo, H., Abe, K., Kitamura, K. and Arai, S. 1996.Oryzacystatins exhibit growth-inhibitory and lethal effects on different species of bean insect pests, Callosobruchus chinensis (Coleoptera) and Riptortus calavatus (Hemiptera).Biosci. Biotechnol. Biochem. 60: 209–212.

    Google Scholar 

  • Laemmli, U.K. 1970.Cleavage of structural proteins during assembly of the head of bacteriophage T4.Nature 277:680–685.

    Google Scholar 

  • Lagrimini, L.M., Burkhart, W., Moyer, M. and Rosthein, S. 1987. Molecular cloning of complementary DNA encoding the lignin forming peroxidases from tobacco: molecular analysis and tissue-specific expression. Proc. Natl. Acad. Sci. USA 84: 7542–7546.

    Google Scholar 

  • Lecardonnel, A., Chauvin, L., Jouanin, L., Beaujean, A., Prevost, G. and Sangwan-Norreel, B. 1999. Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae. Plant Sci.140: 71–79.

    Google Scholar 

  • Li, Z., Sommer, A., Dingermann, T. and Noe, C.R. 1996. Molecular cloning and sequence analysis of a cDNA encoding a cysteine proteinase inhibitor from Sorghum bicolor seedlings.Mol. Gen. Genet. 251:499–502.

    Google Scholar 

  • Lim, C.O., Lee, S.I., Chung, W.S., Park, S.H., Hwang, I. and Cho, M.J. 1996. Characterization of a cDNA encoding a cysteine proteinase inhibitor from chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds.Plant Mol. Biol. 30: 373–379.

    Google Scholar 

  • Margis, R., Reis, E.M. and Villeret, V. 1998.Structural and phylogenetic relationships among plant and animal cystatins.Arch. Biochem. Biophys. 359: 24–30.

    Google Scholar 

  • Mena, M., Vicente-Carbajosa, J., Schmidt, R.J. and Carbonero, P. 1998. An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm.Plant J.16:53–62.

    Google Scholar 

  • Molina A., Díaz, I., Vasil, I.K., Carbonero, P. and García-Olmedo, F. 1996.Two cold inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens.Mol. Gen. Genet.252: 162–168.

    Google Scholar 

  • Ojima, A., Shiota, H., Higashi, K., Kamada, H., Shimma, Y., Wada, M. and Satoh, S. 1997.An extracellular insoluble inhibitor of cysteine proteinases in cell culture and seeds of carrot.Plant Mol. Biol.34:99–109.

    Google Scholar 

  • Pernas, M., Sánchez-Monge, R., Gómez, L. and Salcedo, G. 1998. A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.Plant Mol. Biol.38: 1235–1242.

    Google Scholar 

  • Pernas, M., López-Solanilla, E., Sánchez-Monge, R., Salcedo, G. and Rodriguez-Palenzuela, P. 1999. Antifungal activity of a plant cystatin.Mol. Plant-Microbe Interact.12: 624–627.

    Google Scholar 

  • Pernas, M., Sánchez-Monge, R. and Salcedo, G. 2000. Biotic and abiotic stress can induce cystatin expression in chestnut.FEBS Lett. 467:206–210.

    Google Scholar 

  • Rogers, B.L., Pollock, J., Klapper, D. and Griffith, I.J. 1993. Sequence of the proteinase-inhibitor cystatin homologue from the pollen of Ambrosia artemisiifolia (short ragweed).Gene133: 219–221.

    Google Scholar 

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

    Google Scholar 

  • Schwabe, C., Anastasi A., Crow, H., McDonald, K.J. and Barrett, J.A. 1984. Cystatin: aminoacid sequence and possible secondary structure. Biochem. J.217: 813–817.

    Google Scholar 

  • Song, I., Taylor, M., Baker, K. and Bateman, R.C. 1995. Inhibition of cysteine proteinases by Carica papaya cystatin produced in Escherichia coli.Gene162:221–224.

    Google Scholar 

  • Vain, P., Worland, B., Clarke, M.C., Richard, G., Beavis, M., Liu, H., Kohli, A., Leech, M., Snape, J., Christou, P. and Atkinson, H. 1998.Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-I 1D86) for nematode resistance in transgenic rice plants. Theor. Appl. Genet. 96:266–271.

    Google Scholar 

  • Waldron, C., Wegrich, L.M., Merlo, P.A.O. and Walsch, T.A. 1993. Characterization of a genomic sequence coding for potato multicystatin, an eight domain cysteine proteinase inhibitor.Plant Mol. Biol. 23: 801–812.

    Google Scholar 

  • Zhao, Y., Botella, M.A., Subramanian, L., Niu, X., Nielsen, S.S., Bressan, R.A. and Hasegawa, P.M. 1996. Two wound-inducible soybean cysteine proteinase have greater insect digestive proteinase inhibitory activities than a constitutive homolog.Plant Physiol. 111:1299–1306.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

these authors contributed equally to this work

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gaddour, K., Vicente-Carbajosa, J., Lara, P. et al. A constitutive cystatin-encoding gene from barley (Icy) responds differentially to abiotic stimuli. Plant Mol Biol 45, 599–608 (2001). https://doi.org/10.1023/A:1010697204686

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1010697204686

Navigation