Abstract
Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Current Protocol in Molecular Biology. Greene Publishing Associates/WileyInterscience, New York (1989).
Berkelman T, Houtchens KA, DuPont FM: Two cDNA clones encoding isoforms of the B subunit of the vacuolar ATPase from barley roots. Plant Physiol 104: 287–288 (1994).
Binzel ML, Hess FD, Bressan RA, Hassegawa PM: Intracellular compartmentation of ions in salt adapted tobacco cells. Plant Physiol 86: 607–614 (1988).
Bohnert HJ, Nelson DE, Jensen RG: Adaptations to environmental stresses. Plant Cell 7: 1099–1111 (1995).
Braam J, Davis RW: Rain, wind, and touchinduced expression of calmodulin and calmodulin-related genes. Cell 60: 357–364 (1990).
Cheung WY: Calmodulin plays a pivotal role in cellular regulation. Science 207: 19–26 (1980).
Cunningham KW, Fink GR: Calcineurin inhibits VCX1dependent H+/Ca2+ exchange and induced Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol 16: 2226–2237 (1996).
Davis SJ, Vierstra RD: Soluble derivatives of green fluorescent protein (GFP) for use in Arabidopsis thaliana. Weeds World 3: 43–48 (1996).
Delauney AJ, Verma DPS: Proline biosynthesis and osmoregulation in plants. Plant J 4: 215–223 (1993).
DuPont FM: Salt-induced changes in ion transport: regulation of primary pumps and secondary transporters. In: Cooke DT, Clarkson DT (eds) Transport and Receptor Proteins of Plant Membranes, pp. 91–100. Plenum Press, New York (1992).
Frandsen G, Muller-Uri F, Nielsen M, Mundy J, Skriver K: Novel plant Ca2+binding protein expressed in response to abscisic acid and osmotic stress. J Biol Chem 271: 343–348 (1996).
Garbarino J, DuPont FM: Rapid induction of Na+/H+ exchange activity in barley root tonoplast. Plant Physiol 89: 1–4 (1989).
Gawienowski MC, Szymanski D, Perera IY, Zielinski RE: Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs. Plant Mol Biol 22: 215–225 (1993).
Gossett DR, Banks SW, Millhollon EP, Lucas MC: Antioxidant response to NaCl stress in a control and an NaCl-tolerant cotton cell line grown in the presence of paraquat, buthionine sulfoximine, and exogenous glutathione. Plant Physiol 112: 803–809 (1996).
Han J, Lee JD, Bibbs L, Ulevitch RJ: A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811 (1994).
Hanson AD, Rathinasabapathi B, Rivoal J, Burnet M, Dillon MO, Gage DA: Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance. Proc Natl Acad Sci USA 91: 306–310 (1994).
Hwang I, Goodman HM: An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl. Plant J 8: 37–43 (1995).
Jena PK, Reddy ASN, Poovaiah BW: Molecular cloning and sequencing of a cDNA for plant calmodulin: signal-induced changes in the expression of calmodulin. Proc Natl Acad Sci USA 86: 3644–3648 (1989).
Jonak C, Kiegerl S, Ligterink W, Barker PJ, Huskisson NS, Hirt H: Stress signaling in plants: a mitogenactivated Protein kinase pathway is activated by cold and drought. Proc Natl Acad Sci USA 93: 11274–11279 (1996).
Kolodziej PA, Young RA: Epitope tagging and protein surveillance. Meth Enzymol 194: 508–519 (1991).
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685 (1970).
Lee SH, Kim JC, Lee MS, Heo WD, Seo HY, Yoon HW, Hong JC, Lee SY, Bahk JD, Hwang I, Cho MJ: Identification of a novel divergent calmodulin isoform from soybean which has differential ability to activate calmodulindependent enzymes. J Biol Chem 270: 21806–21812 (1995).
Leung J, Bouvier-Durand M, Morris PC, Guerrier D, Chefdor F, Giraudat J: Arabidopsis ABA response gene ABI1: features of a calciummodulated protein phosphatase. Science 264: 1448–1452 (1994).
Leung J, Merlot S, Giraudat J: The Arabidopsis abscisic acidinsensitive 2 (ABI2) and ABI1 genes encode homologous protein phosphatase 2C involved in abscisic acid signal transduction. Plant Cell 9: 759–771 (1997).
Levine A, Pennell RI, Alvarez ME, Palmer R, Lamb C: Calcium-mediated apoptosis in a plant hypersensitive disease resistance response. Curr Biol 6: 427–437 (1996).
Maeda T, Takekawa M, Saito H: Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science 269: 554–558 (1995).
Maeda T, Wurgler-Murphy SM, Saito H: A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369: 242–245 (1994).
Melnick MB, Melnick C, Lee M, Woodward DO: Structure and sequence of the calmodulin gene from Neurospora crassa. Biochim Biophys Acta 1171: 334–336 (1993).
Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi-Shinozaki K, Matsumoto K, Shinozaki K: A gene encoding a Mitogenactivated protein kinase kinase kinase is induced simultaneously with genes for a mitogenactivated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 93: 765–769 (1996).
Morgan BA, Bouquin N, Johnston LH: Two-component signaltransduction systems in budding yeast MAP a different pathway? Trends Cell Biol 5: 453–457 (1995).
Müller-Röber B, Ellenberg J, Provart N, Willmitzer L, Busch H, Becker D, Dietrich P, Hoth S, Heidrich R: Cloning and electrophysiological analysis of KST, an inward recitifying K+ channel expressed in potato guard cells. EMBO J 14: 2409–2416 (1995).
Nakamura T, Liu Y, Hirata D, Namba H, Harada SI, Hirokawa T, Miyakawa T: Protein phosphatase type 2B (calcineurin)-mediated, FK506-sensitive regulation of intracellular ions in yeast is an important determinant for adaptation to high salt stress conditions. EMBO J 12: 4063–4071 (1993).
Niu X, Bressan RA, Hasegawa PM, Pardo JM: Ion homeostasis in NaCl stress environments. Plant Physiol 109: 735–742 (1995).
Pih KT, Jang HJ, Kang SG, Piao HL, Hwang I: Isolation of molecular markers for salt stress responses in Arabidopsis thaliana. Mol Cell 7: 567–571 (1997).
Poping B, Gibbson T, Watson MD: The Pisum sativum MAP kinase homologue (PsMAPK) rescues the Saccharomyces cerevisiae hog1 deletion mutant under conditions of high osmotic stress. Plant Mol Biol 31, 355–363 (1996).
Posas F, Wurgler-Murphy SM, Maeda T, Witten EA, Thai TC, Saito H: Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1YPDSSK1 ‘twocomponent osmosensor’. Cell 86: 865–875 (1996).
Putkey JA, Slaughter GR, Means AR: Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. J Biol Chem 260: 4704–4712 (1985).
Schachtman DP, Schroeder JI: Structure and transport mechanism of a high affinity potassium uptake transporter from higher plants. Nature 370: 655–658 (1994).
Sheen J: Ca2+-dependent protein kinase and stress signal transduction in plants. Science 274: 1900–1902 (1996).
Singha S, Choudhuri MA: Effect of salinity (NaCl) stress on H2O2 metabolism in Vigna and Oryza seedlings. Biochem Physiol Pflanz 186: 69–74 (1990).
Skriver K, Mundy J: Gene expression in response to abscisic acid and osmotic stress. Plant Cell 2: 503–512 (1990).
Takeuchi Y, Dotson M, Keen NT: Plant transformation: a simple particle bombardment device based on flowing helium. Plant Mol Biol 18: 835–839 (1992).
Walton DC: Biochemistry and physiology of abscisic acid. Annu Rev Plant Physiol 31: 453–489 (1980).
Wieland J, Nitsche AM, Strayle J, Steiner H, Rudolph HK: The PMR2 gene cluster encodes functionally distinct isoforms of a putative Na+ pump in the yeast plasma membrane. EMBO J 14: 3870–3882 (1995).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Jung Jang, H., Tae Pih, K., Gene Kang, S. et al. Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress. Plant Mol Biol 37, 839–847 (1998). https://doi.org/10.1023/A:1006043006211
Issue Date:
DOI: https://doi.org/10.1023/A:1006043006211