PROTON PUMPS AND ANION TRANSPORT IN VITIS-VINIFERA - THE INORGANIC PYROPHOSPHATASE PLAYS A PREDOMINANT ROLE IN THE ENERGIZATION OF THE TONOPLAST

In order to gain insight into vacuolar changes that are associated wit h fruit specialization regarding the accumulation of organic acids, th e properties of tonoplast vesicles extracted from grape cell culture a nd ripe berries were compared. The two primary pumps, PPase (Pyrophosp hatase) and V-ATPase (V-Adenosine Triphosphatase), which were found on both fruit and cell culture tonoplast vesicles, exhibited classical b ehaviour for known inhibitors and activators. However, the 3-fold high er activity of the PPase in comparison to the V-ATPase was unexpected in a fruit exhibiting a vacuolar pH of 3.5. Surprisingly, the hydrolyt ic activities of both pumps were stimulated by temperatures up to 65 d egrees C. Anion transport was studied by measuring simultaneously prot on entry and membrane potential on the two types of tonoplast vesicles . Maximal acidification rates could be observed upon simultaneous ener gization by ATP and PPi, and were abolished by imidodiphosphate and ba filomycin, while vanadate had little effect. Comparison of the sequenc e 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 cultu red cells. The transport of malate and tartrate was completely inhibit ed by 4,4'-diisothio-cyanato-stilbene-2,2'disulphonic acid while chlor ide transport was less affected. Km values for malate and tartrate tra nsports were similar for vesicles extracted from cultured cell and fru its, around 14 mM. The transport rates of malate and tartrate were not additive, suggesting that a similar transporter was involved in the v acuolar uptake of the two major acids of grape berries. (C) Elsevier, Paris.