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Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1

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Abstract

Main conclusion

The Vitis vinifera copper transporter 1 is capable of self-interaction and mediates intracellular copper transport.

Abstract

An understanding of copper homeostasis in grapevine (Vitis vinifera L.) is particularly relevant to viticulture in which copper-based fungicides are intensively used. In the present study, the Vitis vinifera copper transporter 1 (VvCTr1), belonging to the Ctr family of copper transporters, was cloned and functionally characterized. Amino acid sequence analysis showed that VvCTr1 monomers are small peptides composed of 148 amino acids with 3 transmembrane domains and several amino acid residues typical of Ctr transporters. Bimolecular fluorescence complementation (BiFC) demonstrated that Ctr monomers are self-interacting and subcellular localization studies revealed that VvCTr1 is mobilized via the trans-Golgi network, through the pre-vacuolar compartment and located to the vacuolar membrane. The heterologous expression of VvCTr1 in a yeast strain lacking all Ctr transporters fully rescued the phenotype, while a deficient complementation was observed in a strain lacking only plasma membrane-bound Ctrs. Given the common subcellular localization of VvCTr1 and AtCOPT5 and the highest amino acid sequence similarity in comparison to the remaining AtCOPT proteins, Arabidopsis copt5 plants were stably transformed with VvCTr1. The impairment in root growth observed in copt5 seedlings in copper-deficient conditions was fully rescued by VvCTr1, further supporting its involvement in intracellular copper transport. Expression studies in V. vinifera showed that VvCTr1 is mostly expressed in the root system, but transcripts were also present in leaves and stems. The functional characterization of VvCTr-mediated copper transport provides the first step towards understanding the physiological and molecular responses of grapevines to copper-based fungicides.

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Abbreviations

ACT1:

Actin 1

BCS:

Bathocuproine disulfonic acid

BiFC:

Bimolecular fluorescence complementation

CCH:

Copper chaperone

CFP:

Cyan fluorescent protein

CTAB:

Cetyltrimethylammonium bromide

Ctr/COPT:

Copper transporter

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

GFP:

Green fluorescent protein

LB:

Luria–Bertani medium

pCOPT5 :

Promoter region of Arabidopsis COPT5

PIP:

Plasma membrane intrinsic protein

PVP:

Polyvinylpyrrolidone

RAN1:

Responsive to antagonist 1

RFP:

Red fluorescent protein

RRG:

Relative root growth

SC-URA:

Synthetic complete medium without uracil

SNARE:

Syntaxin family of soluble N-ethyl maleimide sensitive factor adaptor protein receptors

TMD:

Transmembrane domain

VvCTr:

Vitis vinifera copper transporter

YFP:

Yellow fluorescent protein

YPEG:

Yeast extract/peptone/ethanol/glycerol medium

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Acknowledgments

We thank Lola Peñarrubia and Antoni Garcia i Molina for providing the Arabidopsis copt5 line used in this study. We are grateful to Dennis J. Thiele for providing the ctr1Δctr3Δctr2Δ and the ctr1Δctr3Δ knock out yeast strains. We thank Niko Geldner for providing the wave line constructs used in this study. We are grateful to Hiromi Tajima for providing the Remorin constructs. This work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Program) - Portuguese Foundation for Science and Technology [FCOMP-01-0124-FEDER-022692, FCOMP-01-0124-FEDER-008760 (Ref. FCT PTDC/AGR-ALI/100636/2008), FCT/5955/27/5/2013/S—scientific cooperation Portugal-Tunisia, PhD grant no. SFRH/BD/64587/2009 to V.M.]; by the networking activities within the European project INNOVINE [ref. 311775]; by the European COST Action [FA1106 “QualityFruit”] and by the Will W. Lester Endowment University of California.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB), Vila Real, Portugal

    Viviana Martins & Hernâni Gerós

  2. Grupo de Investigação em Biologia Vegetal Aplicada e Inovação Agroalimentar—Agrobioplant, Departamento de Biologia, Escola Ciências, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal

    Viviana Martins & Hernâni Gerós

  3. Department of Plant Sciences, University of California, One Shields Ave, Davis, 95616, USA

    Elias Bassil & Eduardo Blumwald

  4. Center of Biotechnology of Borj Cédria, BP 901, 2050, Hammam-Lif, Tunisia

    Mohsen Hanana

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  1. Viviana Martins
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Correspondence to Hernâni Gerós.

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Martins, V., Bassil, E., Hanana, M. et al. Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1. Planta 240, 91–101 (2014). https://doi.org/10.1007/s00425-014-2067-5

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