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Protoplasma

, Volume 256, Issue 1, pp 161–170 | Cite as

Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters

  • Amparo SanzEmail author
  • Sharon Pike
  • Mather A Khan
  • Àngela Carrió-Seguí
  • David G Mendoza-Cózatl
  • Lola Peñarrubia
  • Walter Gassmann
Original Article

Abstract

Copper (Cu) is an essential plant micronutrient. Under scarcity, Cu2+ is reduced to Cu+ and taken up through specific high-affinity transporters (COPTs). In Arabidopsis, the COPT family consists of six members, either located at the plasma membrane (COPT1, COPT2, and COPT6) or in internal membranes (COPT3 and COPT5). Cu uptake by COPT proteins has been mainly assessed through complementation studies in corresponding yeast mutants, but the mechanism of this transport has not been elucidated. To test whether Cu is incorporated by an electrogenic mechanism, electrophysiological changes induced by Cu addition were studied in Arabidopsis thaliana. Mutant (T-DNA insertion mutants, copt2–1 and copt5–2) and overexpressing lines (COPT1OE and COPT5OE) with altered expression of COPT transporters were compared to wild-type plants. No significant changes of the membrane potential (Em) were detected, regardless of genotype or Cu concentration supplied. In contrast, membrane depolarization was detected in response to iron supply in both wild-type and in mutant or transgenic plants. Similar results were obtained for trans-plant potentials (TPP). GFP fusions of the plasma membrane COPT2 and the internal COPT5 transporters were expressed in Xenopus laevis oocytes to potentiate Cu uptake signals, and the cRNA-injected oocytes were tested for electrical currents upon Cu addition using two-electrode voltage clamp. Results with oocytes confirmed those obtained in plants. Cu accumulation in injected oocytes was measured by ICP-OES, and a significant increase in Cu content with respect to controls occurred in oocytes expressing COPT2:GFP. The possible mechanisms driving this transport are discussed in this manuscript.

Keywords

Arabidopsis thaliana Copper uptake COPT transporters Membrane (Em) and trans-plant (TPP) potentials Two-electrode voltage clamp (TEVC) Xenopus laevis oocytes 

Notes

Acknowledgements

This work was performed during a sabbatical leave of AS at the University of Missouri-Columbia. AC-S is recipient of a pre-doctoral fellowship from the Spanish Ministry of Economy, Industry, and Competitiveness. Elemental analyses at UM-C were supported by a US National Science Foundation award (IOS-1252706 to DM-C). We thank the skillful technical help of Li Na Nguyen, Conner Rogan, and Chris Garner (UM-C).

Funding

Travel expenses were financed by the University of Valencia (UV-INV-EPD116-383019) and supported by grants BIO2014-56298-P and BIO2017-87828-C2-1-P (to LP and AS) from the Spanish Ministry of Economy and Competitiveness and FEDER funds from the European Union.

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interests.

Supplementary material

709_2018_1286_MOESM1_ESM.docx (147 kb)
Figure S1 TPP variations in Arabidopsis thaliana WT plants in response to light conditions and induced by addition of 5 mM glucose (Glu) to the medium bathing the roots. Light/dark (L/D) or dark/light (D/L) transitions were applied as indicated in the graph. Numbers in brackets show voltages in mV. (DOCX 146 kb)
709_2018_1286_MOESM2_ESM.docx (1.1 mb)
Figure S2 Confocal images of X. laevis COPT2:GFP (a, c) and COPT5:GFP (b, d) cRNA-injected oocytes showing membrane localization of the green fluorescent signal of GFP (40X magnification). Images were taken 24 h after injection. (DOCX 1129 kb)

References

  1. Andrés-Colás N, Perea-García A, Puig S, Peñarrubia L (2010) Deregulated copper transport affects Arabidopsis development especially in the absence of environmental cycles. Plant Physiol 153:170–184CrossRefGoogle Scholar
  2. Antala S (2016) Molecular insights of the human zinc transporter hZIP4. Dissertation, Worcester Polytechnic InstituteGoogle Scholar
  3. Bernal M, Casero D, Singh V, Wilson GT, Grande A, Yang H, Dodani SC, Pellegrini M, Huijser P, Connolly EM, Merchant SS, Krämer U (2012) Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis. Plant Cell 24:738–761CrossRefGoogle Scholar
  4. Bose J, Babourinal O, Shabala S, Rengel Z (2010) Aluminium-induced ion transport in Arabidopsis: the relationship between Al tolerance and root ion flux. J Exp Bot 61:3163–3175CrossRefGoogle Scholar
  5. Broadly M, Brown P, Cakmak I, Rengel Z, Zhao F (2012) Function of Nutrients: Micronutrients. In: Marschner P (ed) Marschner’s Mineral nutrition in higher plants. Academic Press, Cambridge, pp 191–248CrossRefGoogle Scholar
  6. Chaloupka R, Courville P, Veyrier F, Knudsen B, Tompkins TA, Cellier MFM (2005) Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo. Biochemistry 44:726–733CrossRefGoogle Scholar
  7. De Boer AH, Prins HBA, Zanstra PE (1983) Bi-phasic composition of trans-root electrical potential in roots of Plantago species: involvement of spatially separated electrogenic pumps. Planta 157:259–266CrossRefGoogle Scholar
  8. Durrett TP, Gassmann W, Rogers E (2007) The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiol 144:197–205CrossRefGoogle Scholar
  9. Eppig JJ, Dumont JN (1972) Amino acid pools in developing oocytes of Xenopus laevis. Develop Biol 28:531–536CrossRefGoogle Scholar
  10. Falchuk KH, Montorzi M, Vallee BL (1995) Zinc uptake and distribution in Xenopus laevis oocytes and embryos. Biochemistry 34:16524–16531CrossRefGoogle Scholar
  11. García-Molina A, Andrés-Colás N, Perea-García A, del Valle-Tascón S, Peñarrubia L, Puig S (2011) The intracellular Arabidopsis COPT5 transport protein is required for photosynthetic electron transport under severe copper deficiency. Plant J 65:848–860CrossRefGoogle Scholar
  12. Gayomba SR, Jung H, Yan J, Danku J, Rutzke MA, Bernal M, Krämer U, Kochian LV, Salt DE, Vatamaniuk OK (2013) The CTR/COPT-dependent copper uptake and SPL7-dependent copper deficiency responses are required for basal cadmium tolerance in A. thaliana. Metallomics: integrated biometal. Science 5:1262–1275Google Scholar
  13. Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA (1997) Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature 388:482–488CrossRefGoogle Scholar
  14. Huang N-C, Liu K-H, Lo H-J, Tsay Y-F (1999) Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell 11:1381–1392CrossRefGoogle Scholar
  15. Illes P, Schlicht M, Pavlovkin J, Lichtscheidl I, Baluska F, Ovecka M (2006) Aluminium toxicity in plants: internalization of aluminium into cells of the transition zone in Arabidopsis root apices related to changes in plasma membrane potential, endosomal behaviour and nitric oxide production. J Exp Bot 57:4201–4213CrossRefGoogle Scholar
  16. Jung H, Gayomba SR, Rutzke MA, Craft E, Kochian LV, Vatamaniuk OK (2012) COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter-binding protein-like7. J Biol Chem 287:33252–33267CrossRefGoogle Scholar
  17. Kaempfenkel K, Kushnir S, Babiychuk E, Inzé D, Van Montagu M (1995) Molecular characterization of a putative Arabidopsis thaliana copper transporter and its yeast homologue. J Biol Chem 270:28479–28486CrossRefGoogle Scholar
  18. Kavitha PG, Kuruvilla S, Mathew MK (2015) Functional characterization of a transition metal ion transporter, OsZIP6 from rice (Oryza sativa L.). Plant Physiol Biochem 97:165–174CrossRefGoogle Scholar
  19. Kenderesova L, Stanova A, Pavlovkin J, Durisova E, Nadubinska M, Ciamporova M, Ovecka M (2012) Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species. Ann Bot 110:445–459CrossRefGoogle Scholar
  20. Kennedy CD, Gonsalves FAN (1987) The action of divalent zinc, cadmium, mercury, copper and lead on the trans-root potential and H+ efflux of excised roots. J Exp Bot 38:800–817CrossRefGoogle Scholar
  21. Kennedy CD, Gonsalves FAN (1989) The action of divalent Zn, Cd, Hg, Cu, and Pb ions on the ATPase activity of a plasma membrane fraction isolated from roots of Zea mays. Plant Soil 117:167–175CrossRefGoogle Scholar
  22. Klaumann S, Nickolaus SD, Fürst SH, Starck S, Schneider S, Ekkehard Neuhaus H, Trentmann O (2011) The tonoplast copper transporter COPT5 acts as an exporter and is required for interorgan allocation of copper in Arabidopsis thaliana. New Phytol 192:393–404CrossRefGoogle Scholar
  23. Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2004) OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant J 39:415–424CrossRefGoogle Scholar
  24. Lee J, Peña MMO, Nose Y, Thiele DJ (2002) Biochemical characterization of the human copper transporter Ctr1. J Biol Chem 277:4380–4387CrossRefGoogle Scholar
  25. Lequeux H, Hermans LS, Verbruggen N (2010) Response to copper excess in Arabidopsis thaliana: impact on the root system architecture, hormone distribution, lignin accumulation and mineral profile. Plant Physiol Biochem 48:673–682CrossRefGoogle Scholar
  26. Lin C-M, Kosman DJ (1990) Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae. Kinetics and mechanism. J Biol Chem 265:9194–9200Google Scholar
  27. Llamas A, Ullrich CI, Sanz A (2000) Cd2+ effects on transmembrane electrical potential difference, respiration and membrane permeability of rice (Oryza sativa L) roots. Plant Soil 219:21–28CrossRefGoogle Scholar
  28. Llamas A, Ullrich CI, Sanz A (2008) Ni2+ toxicity in rice: effect on membrane functionality and plant water content. Plant Physiol Biochem 46:905–910CrossRefGoogle Scholar
  29. Ludewig U, von Wiren N, Frommer WB (2002) Uniport of NH4 + by the root hair plasma membrane ammonium transporter LeAMT1;1. J Biol Chem 277:13548–13555CrossRefGoogle Scholar
  30. Manusadzianas L, Maksimov G, Darginaviciene JJ, Jurkoniene S, Sadauskas K, Viktus R (2002) Response of the Charophyte Nitellopsis obtuse to heavy metals at the cellular, cell membrane, and enzyme levels. Environ Toxicol 17:275–283CrossRefGoogle Scholar
  31. Martins V, Hanana M, Blumwald E, Gerós H (2012) Copper transport and compartmentation in grape cells. Plant Cell Physiol 53:1866–1880CrossRefGoogle Scholar
  32. Milner MJ, Seamon J, Craft E, Kochian LV (2013) Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. J Exp Bot 64:369–381CrossRefGoogle Scholar
  33. Montorzi M, Falchuk KH, Vallee BL (1994) Xenopus laevis vitellogenin is a Zn protein. Biochem Biophys Res Comm 200:1407–1413CrossRefGoogle Scholar
  34. Murata Y, Ma JF, Yamaji N, Ueno D, Nomoto K, Iwashita T (2006) A specific transporter for iron(III)-phytosiderophore in barley roots. Plant J 46:563–572CrossRefGoogle Scholar
  35. Murphy AS, Eisinger WR, Shaff JE, Kochian LV, Taiz L (1999) Early copper-induced leakage of K+ from Arabidopsis seedlings is mediated by ion channels and coupled to citrate efflux. Plant Physiol 121:1375–1382CrossRefGoogle Scholar
  36. Nomizu T, Falchuk KH, Vallee BL (1993) Zinc, iron, and copper contents of Xenopus laevis oocytes and embryos. Mol Reprod Dev 36:419–423CrossRefGoogle Scholar
  37. Osawa H, Stacey G, Gassmann W (2006) ScOPT4 function as proton-coupled oligopeptide transporters with broad but distinct substrate specificities. Biochem J 393:267–275Google Scholar
  38. Pavlovkin J, Luxová M, Mistríkova I, Mistrík I (2006) Short- and long-term effects of cadmium on transmembrane electric potential (Em) in maize roots. Biologia 61:109–114CrossRefGoogle Scholar
  39. Peñarrubia L, Romero P, Carrió-Seguí A, Andrés-Bordería A, Moreno J, Sanz A (2015) Temporal aspects of copper homeostasis and its crosstalk with hormones. Front Plant Sci 6:255CrossRefGoogle Scholar
  40. Perea-García A, García-Molina N, Andrés-Colás N, Vera-Sirera F, Pérez-Amador MA, Puig S, Peñarrubia L (2013) Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling. Plant Physiol 162:180–194CrossRefGoogle Scholar
  41. Pike S, Patel A, Stacey G, Gassmann W (2009) Arabidopsis OPT6 is an oligopeptide transporter with exceptionally broad substrate specificity. Plant Cell Physiol 50:1923–1932CrossRefGoogle Scholar
  42. Puig S (2014) Function and regulation of the plant COPT family of the high-affinity copper transport proteins. Advances in Botany 2014:9.  https://doi.org/10.1155/2014/476917
  43. Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV (1999) Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284:805–808CrossRefGoogle Scholar
  44. Ravet K, Pilon M (2013) Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxid Redox Signal 19:919–932CrossRefGoogle Scholar
  45. Reid RJ (2001) Mechanisms of micronutrient uptake in plants. Aust J Plant Physiol 28:659–666Google Scholar
  46. Rodrigo-Moreno A, Poschenrieder C, Shabala S (2013) Transition metals: a double-edge sward in ROS generation and signaling. Plant Sign Behav 8(3):e23425CrossRefGoogle Scholar
  47. Sancenón V, Puig S, Mira H, Thiele DJ, Peñarrubia L (2003) Identification of a copper transporter family in Arabidopsis thaliana. Plant Mol Biol 51:577–587CrossRefGoogle Scholar
  48. Sancenón V, Puig S, Mateu-Andrés I, Dorcey E, Thiele DJ, Peñarrubia L (2004) The Arabidopsis copper transporter COPT1 functions in root elongation and pollen development. J Biol Chem 279:15348–15355CrossRefGoogle Scholar
  49. Sanz A, Llamas A, Ullrich CI (2009) Distinctive phytotoxic effects of Cd and Ni on membrane functionality. Plant Sign Behav 4:980–982CrossRefGoogle Scholar
  50. Schaaf G, Ludewig U, Erenoglu BE, Mori S, Kitahara T, von Wiren N (2004) ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. J Biol Chem 279:9091–9096CrossRefGoogle Scholar
  51. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plant under stressful conditions. J Bot 2012:26.  https://doi.org/10.1155/2012/217037
  52. Sijmons PC, Lanfermeijer FC, De Boer AH, Prins HBA, Bienfait HF (1984) Depolarization of cell membrane potential during trans-plasma membrane electron transfer to extracellular electron acceptors in iron-deficient roots of Phaseolus vulgaris L. Plant Physiol 76:943–946CrossRefGoogle Scholar
  53. Sivaguru M, Pike S, Gassmann W, Baskin TI (2003) Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor. Plant Cell Physiol 44:667–675CrossRefGoogle Scholar
  54. Slayman CL, Slayman CW (1974) Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a proton-dependent cotransport system. Proc Natl Acad Sci U S A 71:1935–1939CrossRefGoogle Scholar
  55. Sobczak K, Bangel-Ruland N, Leier G, Weber W-M (2010) Endogenous transport systems in the Xenopus laevis oocyte plasma membrane. Methods 51:183–189CrossRefGoogle Scholar
  56. Sunderman FW, Plowman MC, Kroftova OS, Grbac-Ivantovic S, Foglia L, Crivello JF (1995) Effects of teratogenic exposures to Zn2+, Cd2+, Ni2+, Co2+, and Cu2+ on metallothionein and metallothionein-mRNA contents of Xenopus embryos. Pharmacol Toxicol 76:178–184CrossRefGoogle Scholar
  57. Tsigelny IF, Sharikov Y, Greenberg JP, Miller MA, Kouznetsova VL, Larson CA, Howell SB (2012) An all-atom model of the structure of human copper transporter 1. Cell Biochem Biophys 63:223–234CrossRefGoogle Scholar
  58. Wegner LH, Sattelmacher B, Läuchli A, Zimmermann U (1999) Trans-root potential, xylem pressure, and root cortical membrane potential of ‘low-salt’ maize plants as influenced by nitrate and ammonium. Plant Cell Environ 22:1549–1558CrossRefGoogle Scholar
  59. Wintz H, Fox T, Wu YY, Feng V, Chen W, Chang HS, Zhu T, Vulpe C (2003) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal novel transporters involved in metal homeostasis. J Biol Chem 278:47644–47653CrossRefGoogle Scholar
  60. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M (2007) Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem 282:16369–16378Google Scholar
  61. Yamasaki H, Hayashi M, Fukazawa M, Kobayashi Y, Shikanai T (2009) SQUAMOSA promoter binding protein-like7 is a central regulator for copper homeostasis in Arabidopsis. Plant Cell 21:347–361CrossRefGoogle Scholar
  62. Zhai Z, Gayomba SR, Jung H, Vimalakumari NK, Piñeros M, Craft E, Rutzke MA, Danku J, Lahner B, Punshon T, Guerinot ML, Salt DE, Kochian LV, Vatamaniuka OK (2014) OPT3 is a phloem-specific iron transporter that is essential for systemic iron signaling and redistribution of iron and cadmium in Arabidopsis. Plant Cell 26:2249–2264CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Dpt de Biologia VegetalUniversitat de ValènciaValenciaSpain
  2. 2.Division of Plant Sciences, CS Bond Life Sciences Center, and Interdisciplinary Plant GroupUniversity of MissouriColumbiaUSA
  3. 3.Dpt de Bioquímica i Biologia Molecular and ERI BiotecmedUniversitat de ValènciaValenciaSpain

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