title: Copper transport and compartmentation in grape cells.
abstract: Copper-based fungicides have been widely used against several grapevine (Vitis vinifera L.) diseases since the late 1800s when the Bordeaux mixture was developed, but their intensive use has raised phytotoxicity concerns. In this study, physiological, biochemical and molecular approaches were combined to investigate the impacts of copper in grape cells and how it is transported and compartmented intracellularly. Copper reduced the growth and viability of grape cells (CSB, Cabernet Sauvignon Berry) in a dose-dependent manner above 100 µM and was accumulated in specific metal ion sinks. The copper-sensitive probe Phen Green SK was used to characterize copper transport across the plasma membrane of CSB cells. The transport system (K(m) = 583 µM; V(max) = 177 × 10(-6) %ΔF min(-1) protoplast(-1)) was regulated by copper availability in the culture medium, stimulated by Ca(2+) and inhibited by Zn(2+). The pH-sensitive fluorescent probe ACMA (9-amino-6-chloro-2-methoxyacridine) was used to evaluate the involvement of proton-dependent copper transport across the tonoplast. Cu(2+) compartmentation in the vacuole was dependent on the transmembrane pH gradient generated by both V-H(+)-ATPase and V-H(+)-pyrophosphatase (PPase). High copper levels in the growth medium did not affect the activity of V-H(+)-PPase but decreased the magnitude of the H(+) gradient generated by V-H(+)-ATPase. Expression studies of VvCTr genes showed that VvCTr1 and VvCTr8 were distinctly affected by CuSO(4) availability in grape cell cultures and that both genes were highly expressed in the green stage of grape berries.
authors.affiliation: Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas, Portugal.
authors.forename: Viviana
authors.initials: V
authors.lastname: Martins
authors.forename: Mohsen
authors.initials: M
authors.lastname: Hanana
authors.forename: Eduardo
authors.initials: E
authors.lastname: Blumwald
authors.forename: Hernâni
authors.initials: H
authors.lastname: Gerós
mesh_terms: D000609:Aminoacridines / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival; D003300:Copper / Q000378:metabolism*; D019327:Copper Sulfate / Q000494:pharmacology; D003470:Culture Media / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005638:Fruit / Q000166:cytology / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000378:metabolism; D059828:Plant Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D027843:Vitis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D000609:Aminoacridines; D003470:Culture Media; D010940:Plant Proteins; D017494:Proton Pumps; C010847:9-amino-6-chloro-2-methoxyacridine; D003300:Copper; D025262:Vacuolar Proton-Translocating ATPases; D043564:Inorganic Pyrophosphatase; D019327:Copper Sulfate