Copper uptake mechanism of Arabidopsis thaliana high-affinity COPT transporters
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.
KeywordsArabidopsis thaliana Copper uptake COPT transporters Membrane (Em) and trans-plant (TPP) potentials Two-electrode voltage clamp (TEVC) Xenopus laevis oocytes
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).
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.
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Conflict of interests
The authors declare that they have no conflict of interests.
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