FURTHER EVIDENCE FOR STACHYOSE AND SUCROSE/H+ ANTIPORTERS ON THE TONOPLAST OF JAPANESE ARTICHOKE (STACHYS-SIEBOLDII) TUBERS

Vacuoles of Japanese artichoke (Stachys sieboldii) tubers accumulate up to 180 mm stachyose (alpha-galactose-[l --> 6]-alpha-galactose-[1 --> 6]-alpha-glucose-[1 <-- --> 2]-beta-fructose) against a concentration gradient, probably by means of an active stachyose/H+ antiporter situated on the tonoplast. The goal of this study was to use isolated tonoplast vesicles to provide further evidence for the existence of such a transport mechanism. Therefore, vesicles were prepared from purified vacuoles of dormant tubers. ATP- and pyrophosphate (PPi)-dependent fluorescence quenching of the DELTApH probe 9-amino-6-chloro-2-methoxyacridine (ACMA) indicated that these vesicles were capable of building up a pH gradient (DELTApH, inside acid). The potent V-type H+-ATPase inhibitor bafilomycin prevented the formation of a DELTApH in the vesicles. Bafilomycin (as well as nitrate, but not vanadate) also inhibited ATP hydrolysis, confirming the tonoplast origin of the isolated vesicles. Addition of stachyose (or sucrose, but not of mannitol) to energized vesicles caused a recovery of ACMA fluorescence, indicating a sugar-dependent dissipation of DELTApH. The rate of fluorescence recovery was dependent on the external sugar concentration used. It displayed a single saturable response to increasing sugar concentrations. Apparent K(m) values of 52 and 25 mm were computed for stachyose and sucrose antiporter activities, respectively. It was also demonstrated that energized vesicles showed a much higher rate of [C-14]stachyose (3 mm) and [C-14]sucrose (i mm) uptake than deenergized vesicles. The results obtained with isolated tonoplast vesicles were very similar to those obtained earlier with intact vacuoles and, therefore, confirm the existence of active stachyose and sucrose/H+ antiporters on the tonoplast of Stachys tuber vacuoles.