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Molecular cloning and characterization of the chloride channel gene family in trifoliate orange

  • Original Papers
  • Published:
Biologia Plantarum

Abstract

Chloride channels (CLCs) play pivotal roles in plant development and anion transport. However, little research has been conducted about the CLC in fruit-bearing plants. Here we provide an insight into the evolution and expression patterns of CLC gene family members in various tissues of trifoliate orange [Poncirus trifoliata (L.) Raf.] and their responses to several treatments. Genome-wide analysis identified six PtrCLC genes. The predicted proteins had similar numbers of amino acids, but shared a low sequence identity. Phylogenetic analysis revealed that PtrCLC were classified into two separate subgroups, and PtrCLC4 and PtrCLC6 in subgroup II were more closely related to bacterial CLCs. Sequence comparison with EcCLCA from Escherichia coli reveals that PtrCLC showed amino acid divergence in anion selectivity of CLC proteins. Real time qPCR analysis shows that PtrCLC genes, particularly PtrCLC6, preferentially expressed in leaves. Nitrogen deficiency irreversibly inhibited expression of PtrCLC genes except for PtrCLC1. In contrast, NaCl stress profoundly induced expression of PtrCLC genes, particularly PtrCLC2 and PtrCLC4, both of which were also upregulated by ABA treatment. The results presented here provide a solid foundation for a future functional research on citrus CLC genes.

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Abbreviations

CBS:

cystathionine β-synthase

CLC:

chloride channel

PtrCLC:

CLC of Poncirus trifoliata

RT-qPCR:

real time quantitative PCR

TM:

trans-membrane regions

References

  • Basilio, D., Noack, K., Picollo, A., Accardi, A.: Conformational changes required for H+/Cl− exchange mediated by a CLC transporter. — Nat. struct. mol. Biol. 21: 456–463, 2014.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bergsdorf, E.Y., Zdebik, A.A., Jentsch, T.J.: Residues important for nitrate/proton coupling in plant and mammalian CLC transporters. — J. biol. Chem. 284: 11184–11193, 2008.

    Article  Google Scholar 

  • Cutler, S.R., Rodriguez, P.L., Finkelstein, R.R., Abrams, S.R.: Abscisic acid: emergence of a core signaling network. — Annu. Rev. Plant Biol. 61: 651–679, 2010.

    Article  CAS  PubMed  Google Scholar 

  • De Angeli, A., Monachello, D., Ephritikhine, G., Frachisse, J.M., Thomine, S., Gambale, F., Barbier-Brygoo, H.: The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. — Nature 442: 939–942, 2006.

    Article  PubMed  Google Scholar 

  • De Angeli, A., Monachello, D., Ephritikhine, G., Frachisse, J.M., Thomine, S., Gambale, F., Barbier-Brygoo, H.: CLCmediated anion transport in plant cells. — Philos. Trans. roy. Soc. B-biol. Sci. 364: 195–201, 2009.

    Article  Google Scholar 

  • Diedhiou, C., Golldack, D.: Salt-dependent regulation of chloride channel transcripts in rice. — Plant Sci. 170: 793–800, 2005.

    Article  Google Scholar 

  • Dutzler, R.: The CLC family of chloride channels and transporters. — Curr. Opin. Struct. Biol. 16: 439–446, 2006.

    Article  CAS  PubMed  Google Scholar 

  • Dutzler, R., Campbell, E.B., Cadene, M., Chait, B.T., MacKinnon, R.: X-ray structure of a ClC chloride channel at 3.0 A reveals the molecular basis of anion selectivity. — Nature 415: 287–294, 2002.

    Article  CAS  PubMed  Google Scholar 

  • Gaxiola, R.A., Yuan, D.S., Klausner, R.D., Fink, G.R.: The yeast CLC chloride channel functions in cation homeostasis. — Proc. nat. Acad. Sci. USA 95: 4046–4050, 1998.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Geelen, D., Lurin, C., Bouchez, D., Frachisse, J.M., Lelièvre, F., Courtial, B., Barbier-Brygoo, H., Maurel, C.: Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content. — Plant J. 21: 259–267, 2000.

    Article  CAS  PubMed  Google Scholar 

  • Harada, H., Kuromori, T., Hirayama, T., Shinozaki, K., Leigh, R.A.: Quantitative trait loci analysis of nitrate storage in Arabidopsis leading to an investigation of the contribution of the anion channel gene, AtCLC-c, to variation in nitrate levels. — J. exp. Bot. 55: 2005–2014, 2004.

    Article  CAS  PubMed  Google Scholar 

  • Hechenberger, M., Wolf N Fischer, B.S., Frommer, W.B., Jentsch, T.J., Steinmeyer, K.: A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption. — J. biol. Chem. 271: 33632–33638, 1996.

    Article  CAS  PubMed  Google Scholar 

  • Jentsch, T.J.: CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. — Crit. Rev. Biochem. mol. Biol. 43: 3–36, 2008.

    Article  CAS  PubMed  Google Scholar 

  • Jentsch, T.J., Neagoe, I., Scheel, O.: CLC chloride channels and transporters. — Curr. Opin. Neurobiol. 15: 319–325, 2005.

    Article  CAS  PubMed  Google Scholar 

  • Jossier, M., Kroniewicz, L., Dalmas, F., Le Thiec, D., Ephritikhine, G., Thomine, S., Barbier-Brygoo, H., Vavasseur, A., Filleur, S., Leonhardt, N.: The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance. — Plant J. 64: 563–576, 2010.

    Article  CAS  PubMed  Google Scholar 

  • Li, W.Y.F., Wong, F.L., Tsai, S.N., Phang, T.H., Shao, G.H., Lam, H.M.: Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells. — Plant Cell Environ. 29: 1122–1137, 2006.

    Article  CAS  PubMed  Google Scholar 

  • Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J., Maurel, C.: Cloning and functional expression of a plant voltagedependent chloride channel. — Plant Cell 8: 701–711, 1996.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lv, Q.D., Tang, R.J., Liu, H., Gao, X.S., Li, Y.Z., Zheng, H.Q., Zhang, H.X.: Cloning and molecular analyses of the Arabidopsis thaliana chloride channel gene family. — Plant Sci. 176: 650–661, 2009.

    Article  CAS  Google Scholar 

  • Marmagne, A., Vinauger-Douard, M., Monachello, D., De Longevialle, A.F., Charon, C., Allot, M., Rappaport, F., Wollman, F.A., Barbier-Brygoo, H., Ephritikhine, G.: Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively. — J. exp. Bot. 58: 3385–3393, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Matulef, K., Maduke, M.: The CLC ‘chloride channel’ family: revelations from prokaryotes. — Mol. Membr. Biol. 24: 342–350, 2007.

    Article  CAS  PubMed  Google Scholar 

  • Monachello, D., Allot, M., Oliva, S., Krapp, A., Daniel-Vedele, F., Barbier-Brygoo, H., Ephritikhine, G.: Two anion transporters AtClCa and AtClCe fulfil interconnecting but not redundant roles in nitrate assimilation pathways. — New Phytol. 183: 88–94, 2009.

    Article  CAS  PubMed  Google Scholar 

  • Nakamura, A., Fukuda, A., Sakai, S., Tanaka, Y.: Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.). — Plant Cell Physiol. 47: 32–42, 2006.

    Article  CAS  PubMed  Google Scholar 

  • Pusch, M.: Structural insights into chloride and proton-mediated gating of CLC chloride channels. — Biochemistry 43: 1135–1144, 2004.

    Article  CAS  PubMed  Google Scholar 

  • Robinson, N.C., Huang, P., Kaetzel, M.A., Lamb, F.S., Nelson, D.J.: Identification of an N-terminal amino acid of the CLC-3 chloride channel critical in phosphorylation-dependent activation of a CaMKII-activated chloride current. — J. Physiol. 556: 353–368, 2004.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Schmittgen, T.D., Livak, K.J.: Analyzing real-time PCR data by the comparative CT method. — Natur. Protocols 3: 1101–1108, 2008.

    Article  CAS  Google Scholar 

  • Teakle, N.L., Tyerman, S.D.: Mechanisms of Cl- transport contributing to salt tolerance. — Plant Cell Environ. 33: 566–589, 2010.

    Article  CAS  PubMed  Google Scholar 

  • Tregeagle, J.M., Tisdall, J.M., Tester, M., Walker, R.R.: Cluptake, transport and accumulation in grapevine rootstocks of differing capacity for Cl—exclusion. — Funct. Plant Biol. 37: 665–673, 2010.

    Article  CAS  Google Scholar 

  • Von der Fecht-Bartenbach, J., Bogner, M., Dynowski, M., Ludewig, U.: CLC-b-mediated NO3 −/H+ exchange across the tonoplast of Arabidopsis vacuoles. — Plant Cell Physiol. 51: 960–968, 2010.

    Article  PubMed  Google Scholar 

  • Von der Fecht-Bartenbach, J., Bogner, M., Krebs, M., Stierhof, Y.D., Schumacher, K., Ludewig, U.: Function of the anion transporter AtCLC-d in the trans-Golgi network. — Plant J. 50: 466–474, 2007.

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang, S., Su, S.Z., Wu, Y., Li, S.P., Shan, X.H., Liu, H.K., Wang, S., Yuan, Y.P.: Overexpression of maize chloride channel gene Zm-CLC-d in Arabidopsis thaliana improved its stress resistance. — Biol. Plant 59: 55–64, 2015.

    Article  CAS  Google Scholar 

  • Wang, Y.Y., Hsu, P.K., Tsay, Y.F.: Uptake, allocation and signaling of nitrate. — Trends Plant Sci. 17: 458–467, 2012.

    Article  CAS  PubMed  Google Scholar 

  • Wege, S., Jossier, M., Filleur, S., Thomine, S., Barbier-Brygoo, H., Gambale, F., De Angeli, A.: The proline 160 in the selectivity filter of the Arabidopsis NO3 −/H+ exchanger AtCLCa is essential for nitrate accumulation in planta. — Plant J. 63: 861–869, 2010.

    Article  CAS  PubMed  Google Scholar 

  • Wei, Q.J., Liu, Y.Z., Zhou, G.F., Li, Q.H., Yang, C.Q., Peng, S.A.: Overexpression of CsCLCc, a chloride channel gene from poncirus trifoliata, enhances salt tolerance in Arabidopsis. — Plant mol. Biol. Rep. 31: 1–10, 2013.

    Article  CAS  Google Scholar 

  • Wong, T.H., Li, M.W., Yao, X.Q., Lam, H.M.: The GmCLC1 protein from soybean functions as a chloride ion transporter. — J. Plant Physiol. 170: 101–104, 2013.

    Article  CAS  PubMed  Google Scholar 

  • Xu, Q., Chen, L.L., Ruan, X., Chen, D., Zhu, A., Chen, C., Bertrand, D., Jiao, W.B., Hao, B.H., Lyon, M.P., et al.: The draft genome of sweet orange (Citrus sinensis). — Nat. Genet. 45: 59–66, 2013.

    Article  CAS  PubMed  Google Scholar 

  • Zhou, G.A., Qiu, L.J.: Identification and functional analysis on abiotic stress response of soybean Cl− channel gene GmCLCnt. — Agr. Sci. China 9: 199–206, 2010.

    Article  CAS  Google Scholar 

  • Zifarelli G, Pusch M.: CLC transport proteins in plants. — Febs Lett. 584: 2122–2127, 2010.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, P.R. China

    Q. J. Wei, N. N. Wang, C. Q. Yang & S. A. Peng

  2. College of Agronomy, Jiangxi Agricultural University, Nanchang, 330045, P.R. China

    Q. J. Wei & Q. Q. Gu

Authors
  1. Q. J. Wei
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  2. Q. Q. Gu
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  3. N. N. Wang
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  4. C. Q. Yang
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  5. S. A. Peng
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Correspondence to S. A. Peng.

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Acknowledgements: This research was financially supported by the Earmarked Fund of China Agriculture Research Systems (CARS-27) and the National Natural Science Foundation of China (grant No. 31460496).

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Wei, Q.J., Gu, Q.Q., Wang, N.N. et al. Molecular cloning and characterization of the chloride channel gene family in trifoliate orange. Biol Plant 59, 645–653 (2015). https://doi.org/10.1007/s10535-015-0532-z

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  • DOI: https://doi.org/10.1007/s10535-015-0532-z

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