Proton pumps and anion transport in Vitis vinifera: The inorganic pyrophosphatase plays a predominant role in the energization of the tonoplast

Abstract

In order to gain insight into vacuolar changes that are associated with fruit specialization regarding the accumulation of organic acids, the properties of tonoplast vesicles extracted from grape cell culture and ripe berries were compared. The two primary pumps, PPase (Pyrophosphatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture tonoplast vesicles, exhibited classical behaviour for known inhibitors and activators. However, the 3-fold higher activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolytic activities of both pumps were stimulated by temperatures up to 65 °C. Anion transport was studied by measuring simultaneously proton entry and membrane potential on the two types of tonoplast vesicles. Maximal acidification rates could be observed upon simultaneous energization by ATP and PPi, and were abolished by imidodiphosphate and bafilomycin, while vanadate had little effect. Comparison of the sequence of transport rates observed with mineral and organic anions on fruit or cultured cell tonoplast vesicles suggested that the fruit vacuole was more specialized in the transport of organic anions than the cultured cells. The transport of malate and tartrate was completely inhibited by 4,4′-diisothio-cyanato-stilbene-2,2′disulphonic acid while chloride transport was less affected. Km values for malate and tartrate transports were similar for vesicles extracted from cultured cell and fruits, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the vacuolar uptake of the two major acids of grape berries.