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.

Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis

“…The Arabidopsis CTr1 and CTr2 (Sancenón et al 2003) and all rice CTr genes (Yuan et al 2011), which code for plasma membrane proteins, are repressed by high copper concentrations. There are eight genes encoding putative V. vinifera copper transporters (VvCTrs) in the grapevine genome (Martins et al 2012). Different from Arabidopsis and rice, the expression of CTr1 and CTr2 in grapevine was induced at high copper concentrations in our study, and the result was similar with Martins et al (2012) who reported that VvCTr1 expression increased after prolonged copper exposure (6 days) or after a short copper pulse (8 h) in CSB cells.…”

“…There are eight genes encoding putative V. vinifera copper transporters (VvCTrs) in the grapevine genome (Martins et al 2012). Different from Arabidopsis and rice, the expression of CTr1 and CTr2 in grapevine was induced at high copper concentrations in our study, and the result was similar with Martins et al (2012) who reported that VvCTr1 expression increased after prolonged copper exposure (6 days) or after a short copper pulse (8 h) in CSB cells. In addition, VvCTr5 was poorly detected in two studies.…”

“…Cu must be released from the root vacuole for long-distance transport to aerial organs (Garcia-Molina et al 2011;Klaumann et al 2011). There are eight genes encoding putative Vitis vinifera copper transporters (VvCTrs) in the grapevine genome (Martins et al 2012). The studies showed that VvCTr1 is mostly expressed in the root system than leaves and stems in V. vinifera.…”

Gli1 contributes to cellular resistance to cisplatin through altered cellular accumulation of the drug

“…This has been reviewed by Hall et al (8). Many of these genes have been shown to be highly conserved, with analogs that exist in grapes (29), rice (30), barley (31) and a range of vertebrates. In humans, these genes tend to be highly expressed in kidney, in the inner ear, and in various tissues throughout the body (8).…”

Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport

“…The membrane pellet was resuspended in 0.3 to 1 mL of resuspension buffer, frozen with liquid nitrogen, and kept at 280°C until use. Tonoplast vesicles from grape (Vitus vinifera) cell cultures were isolated according to Queirós et al (2009) with the modifications from Martins et al (2012). Protein quantification was performed using the method described by Lowry et al (1951).…”