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Cloning and Characterization of a Ca2+/H+ Antiporter from Halophyte Suaeda salsa L.

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Abstract

We have isolated the cDNA of Ca2+/H+ antiporter which was designated as Suaeda salsa cation exchanger 1 (SsCAX1) from the C3 halophyte S. salsa L. To ascertain the location of SsCAX1 in the cell, a peptide-specific antibody to SsCAX1 was prepared, and Western blotting analysis showed that it reacted only with a 48.8-kDa protein from S. salsa vacuolar membrane. Furthermore, SsCAX1 could resume yeast vacuolar Ca2+ transport mutants growth in high Ca2+ concentration (200 mM) culture medium. Northern blotting analysis showed that SsCAX1 expression was mainly found in the leaves and stems and slightly in the roots of S. salsa seedlings. Moreover, SsCAX1 expression levels and the protein amounts were significantly upregulated by CaCl2 and NaCl treatment, respectively. In addition, the upregulation of the expression levels of V-H+-ATPase subunit c coordinated with the expression levels of the Ca2+/H+ antiporter under salinity. These results suggested that SsCAX1 from halophyte S. salsa might be a Ca2+ transporter at tonoplast and plays a key role in maintaining cytosolic Ca2+ homeostasis under saline condition.

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Abbreviations

CAX:

Cation exchanger

DTT:

Dithiothreitol

EGTA:

Ethylene glycol bis(β-aminoethyl)tetraacetic acid

HEPES:

N-2-hydroxyethyl-piperazine-N′-2-ethanesulfonic acid

MES:

2-(N-morpholine)ethanesulfonic acid

PMSF:

Phenylmethyl sulfonyl fluoride

PVP:

Polyvinylpyrrolidone

Tricine:

N-tris-(hydroxymethyl)-methylglycine

Tris:

Tris(hydroxymethyl)aminomethane

References

  • Barkla BJ, Hirschi KD, Pittman JK (2008) Exchangers man the pumps: functional interplay between proton pumps and proton-coupled Ca2+ exchangers. Plant Signal Behav 3:354–356

    Article  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Cheng NH, Pittman JK, Shigaki T, Hirschi KD (2002) Characterization of CAX4, an Arabidopsis H+/cation antiporter. Plant Physiol 128:1245–1254

    Article  PubMed  CAS  Google Scholar 

  • Cheng NH, Pittman JK, Zhu JK, Hirschi KD (2004) The protein kinase SOS2 activates the Arabidopsis H+/Ca2+ antiporter CAX1 to integrate calcium transport and salt tolerance. J Biol Chem 279:2922–2926. doi:10.1074/jbc.M309084200

    Article  PubMed  CAS  Google Scholar 

  • Chomczynski P, Sacci N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159

    Article  PubMed  CAS  Google Scholar 

  • Cunningham KW, Fink GR (1996) Calcineurin inhibits VCX1-dependent H+/Ca2+ exchange and induces Ca2+ ATPases in Saccharomyces cerevisiae. Mol Cell Biol 16:2226–2237

    PubMed  CAS  Google Scholar 

  • Edelist C, Raffoux X, Falque M, Dillmann C, Sicard D, Rieseberg LH, Karrenberg S (2009) Differential expression of candidate salt-tolerance genes in the halophyte Helianthus paradoxus and its glycophyte progenitors H. annuus and H. petiolaris (Asteraceae). Am J Bot 96:1830–1838. doi:10.3732/ajb.0900067

    Article  CAS  Google Scholar 

  • Edmond C, Shigaki T, Ewert S, Nelson MD, Connorton JM, Chalova V, Noordally Z, Pittman JK (2009) Comparative analysis of CAX2-like cation transporters indicates functional and regulatory diversity. Biochem J 418:145–154. doi:10.1042/BJ20081814

    Article  PubMed  CAS  Google Scholar 

  • Evans NH, McAinsh MR, Hetherington AM (2001) Calcium oscillations in higher plants. Curr Opin Plant Biol 4:415–420

    Article  PubMed  CAS  Google Scholar 

  • Geisler M, Frangne N, Gomès E, Martinoia E, Palmgren MG (2000) The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast. Plant Physiol 124:1814–1827

    Article  PubMed  CAS  Google Scholar 

  • Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the mechanism of high efficiency transformation of intact yeast cells. Yeast 11:355–360

    Article  PubMed  CAS  Google Scholar 

  • Han N, Shao Q, Lu CM, Wang BS (2005) The leaf tonoplast V-H+-ATPase activity of a C3 halophyte Suaeda salsa is enhanced by salt stress in a Ca-dependent mode. J Plant Physiol 162:267–274. doi:10.1016/j.jplph.2004.07.016

    Article  PubMed  CAS  Google Scholar 

  • Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499

    Article  PubMed  CAS  Google Scholar 

  • Hirschi KD, Zhen RG, Cunningham KW, Rea PA, Fink GR (1996) CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proc Natl Acad Sci 93:8782–8786

    Article  PubMed  CAS  Google Scholar 

  • Hirschi KD, Korenkov VD, Wilganowski NL, Wagner GJ (2000) Expression of Arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance. Plant Physiol 124:125–133

    Article  PubMed  CAS  Google Scholar 

  • Kamiya T, Maeshima M (2004) Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations. J Biol Chem 279:812–819. doi:10.1074/jbc.M309726200

    Article  PubMed  CAS  Google Scholar 

  • Kamiya T, Akahori T, Maeshima M (2005) Expression profile of the genes for rice cation/H+ exchanger family and functional analysis in yeast. Plant Cell Physiol 46:1735–1740. doi:10.1093/pcp/pci173

    Article  PubMed  CAS  Google Scholar 

  • Kaplan B, Davydov O, Knight H, Galon Y, Knight MR, Fluhr R, Fromm H (2006) Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. Plant Cell 18:2733–2748. doi:10.1105/tpc.106.042713

    Article  PubMed  CAS  Google Scholar 

  • Knight H, Knight MR (2000) Imaging spatial and cellular characteristics of low temperature calcium signature after cold acclimation in Arabidopsis. J Exp Bot 51:1679–1686

    Article  PubMed  CAS  Google Scholar 

  • Koren'kov V, Park S, Cheng NH, Sreevidya C, Lachmansingh J, Morris J, Hirschi KD, Wagner GJ (2007) Enhanced Cd2+-selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers. Planta 225:403–411. doi:10.1007/s00425-006-0352-7

    Article  PubMed  Google Scholar 

  • Lu CM, Qiu NW, Wang BS, Zhang JH (2003) Salinity treatment shows no effects on photosystem II photochemistry, but increases the resistance of photosystem II to heat stress in halophyte Suaeda salsa. J Exp Bot 54:851–860

    Article  PubMed  CAS  Google Scholar 

  • Mandala S, Taiz L (1985) Partial purification of a tonoplast ATPase from corn coleoptiles. Plant Physiol 78:327–333

    Article  PubMed  CAS  Google Scholar 

  • Oettinger HF, Pasqualini R, Bernfield M (1992) Recombinant peptides as immunogens: a comparison of protocols for antisera production using the pGEX system. Biotechniques 12:544–549

    PubMed  CAS  Google Scholar 

  • Pittman JK, Hirschi KD (2001) Regulation of CAX1, an Arabidopsis Ca2+/H+ antiporter: identification of an N-terminal autoinhibitory domain. Plant Physiol 127:1020–1029

    Article  PubMed  CAS  Google Scholar 

  • Pittman JK, Shigaki T, Cheng NH, Hirschi KD (2002a) Mechanism of N-terminal autoinhibition in the Arabidopsis Ca2+/H+ antiporter CAX1. J Biol Chem 277:26452–26459

    Article  PubMed  CAS  Google Scholar 

  • Pittman JK, Sreevidya CS, Shigaki T, Ueoka-Nakanishi H, Hirschi KD (2002b) Distinct N-terminal regulatory domains of Ca2+/H+ antiporters. Plant Physiol 130:1054–1062

    Article  PubMed  CAS  Google Scholar 

  • Qiu NW, Chen M, Guo JR, Ma XL, Wang BS (2007) Coordinate up-regulation of V-H+-ATPase and vacuolar Na+/H+ antiporter as a response to NaCl treatment in a C3 halophyte Suaeda salsa. Plant Sci 172:1218–1225. doi:10.1016/j.plantsci.2007.02.013

    Article  CAS  Google Scholar 

  • Qudeimat E, Frank W (2009) Ca2+ signatures: the role of Ca2+-ATPases. Plant Signal Behav 4:350–352

    Article  PubMed  CAS  Google Scholar 

  • Ranf S, Wünnenberg P, Lee J, Becker D, Dunkel M, Hedrich R, Scheel D, Dietrich P (2008) Loss of the vacuolar cation channel, AtTPC1, does not impair Ca2+ signals induced by abiotic and biotic stresses. Plant J 53:287–299

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sanders D, Pelloux J, Brownlee C, Harper JF (2002) Calcium at the crossroads of signaling. Plant Cell 14(Suppl):401–417

    Google Scholar 

  • Shigaki T, Cheng NH, Pittman JK, Hirschi KD (2001) Structural determinants of Ca2+ transport in the Arabidopsis Ca2+/H+ antiporter CAX. J Biol Chem 276:43152–43159

    Article  PubMed  CAS  Google Scholar 

  • Shigaki T, Pittman JK, Hirschi KD (2003) Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2. J Biol Chem 278:6610–6617. doi:10.1074/jbc.M209952200

    Article  PubMed  CAS  Google Scholar 

  • Shigaki T, Rees I, Nakhleh L, Hirschi KD (2006) Identification of three distinct phylogenetic groups of CAX cation/proton antiporters. J Mol Evol 63:815–825. doi:10.1007/s00239-006-0048-4

    Article  PubMed  CAS  Google Scholar 

  • Sze H, Li X, Palmgren MG (1999) Energization of plant cell membranes by H+-pumping ATPase: regulation and biosynthesis. Plant Cell 11:677–689

    Article  PubMed  CAS  Google Scholar 

  • Ueoka-Nakanishi H, Tsuchiya T, Sasaki M, Nakanishi Y, Cunningham KW, Maeshima M (2000) Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells. Eur J Biochem 267:3090–3098

    Article  PubMed  CAS  Google Scholar 

  • Waditee R, Hossain GS, Tanaka S, Nakamura T, Shikata M, Takano J, Takabe T (2004) Isolation and functional characterization of Ca2+/H+ antiporters from Cyanobacteria. J Biol Chem 279:4330–4338. doi:10.1074/jbc.M310282200

    Article  PubMed  CAS  Google Scholar 

  • Wang BS, Lüttge U, Ratajczak R (2001) Effect of salt treatment and osmotic stress on V-ATPase and V-PPase in leaves of the halophyte Suaeda salsa. J Exp Bot 52:2355–2365

    Article  PubMed  CAS  Google Scholar 

  • Zhao J, Barkla BJ, Marshall J, Pittman JK, Hirschi KD (2008) The Arabidopsis cax3 mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H+-ATPase activity. Planta 227:659–669

    Article  PubMed  CAS  Google Scholar 

  • Zhao J, Connorton JM, Guo Y, Li X, Shigaki T, Hirschi KD, Pittman JK (2009) Functional studies of split Arabidopsis Ca2+/H+ exchangers. J Biol Chem 284:34075–34083. doi:10.1074/jbc.M109.070235

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful for the financial support from the National Natural Science Research Foundation of China (project nos. 30870138 and 30670177), the HI-Tech Research and Development (863) Program of China (2007AA091701), and the Doctoral Foundation of Shandong Province (BS2009NY035).We also thank Professor Dr. Kendal D. Hirschi (Baylor College of Medicine, USA) for providing S. cerevisiae strain K667.

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Authors and Affiliations

  1. Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, No.88, East Wenhua Road, Jinan, 250014, Shandong Province, People’s Republic of China

    Ning Han, Qun Shao, Huayin Bao & Baoshan Wang

  2. College of Food and Biologic Engineering, Shandong Institute of Light Industry, Jinan, 250353, People’s Republic of China

    Ning Han

  3. College of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, 250353, People’s Republic of China

    Huayin Bao

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  1. Ning Han
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  2. Qun Shao
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Correspondence to Baoshan Wang.

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Ning Han and Qun Shao made equal contributions to this work.

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Han, N., Shao, Q., Bao, H. et al. Cloning and Characterization of a Ca2+/H+ Antiporter from Halophyte Suaeda salsa L.. Plant Mol Biol Rep 29, 449–457 (2011). https://doi.org/10.1007/s11105-010-0244-7

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