Uptake of salicylic acid 2-O-β-D-glucose into soybean tonoplast vesicles by an ATP-binding cassette transporter-type mechanism

In soybean (Glycine max L.), salicylic acid (SA) is converted primarily to SA 2-O-beta-D-glucose (SAG) in the cytoplasm and then accumulates exclusively in the vacuole. However, the mechanism involved in the vacuolar transport of SAG has not been investigated. The vacuolar transport of SAG was characterized by measuring the uptake of [C-14]SAG into tonoplast vesicles isolated from etiolated soybean hypocotyls. The uptake of SAG was stimulated about six-fold when MgATP was included in the assay media. In contrast, the uptake of SA was only stimulated 1.25-fold by the addition of MgATP and was 2.2-fold less than the uptake of SAG providing an indication that the vacuolar uptake of SA is promoted by glucosylation. The ATP-dependent uptake of SAG was inhibited by increasing concentrations of vanadate (64% inhibition in the presence of 500 muM) but was not very sensitive to inhibition by bafilomycin A(1) (a specific inhibitor of vacuolar H+-ATPase; EC 3.6.1.3), and dissipation of the transtonoplast H+-electrochemical gradient. The SAG uptake exhibited Michaelis-Menten-type saturation kinetics with a K-m value of 90 muM for SAG. SAG uptake was inhibited 60% by beta-estradiol 17-(beta-D-glucuronide), but glutathione conjugates and uncharged glucose conjugates were only slightly inhibitory. Based on the characteristics of SAG uptake into soybean tonoplast vesicles it is likely that this uptake occurs through an ATP-binding cassette transporter-type mechanism. However, this vacuolar uptake mechanism is not universal since the uptake of SAG by red beet (Beta vulgaris L) tonoplast vesicles appears to involve an H+-antiport mechanism.