The metabolism of salicylic acid (SA) in tobacco (Nicotiana tabacum L. cv. KY 14) cell suspension cultures was examined by adding [7-14C]SA to the cell cultures for 24 h and identifying the metabolites through high performance liquid chromatography analysis. The three major metabolites of SA were SA 2-O-beta-D: -glucose (SAG), methylsalicylate 2-O-beta-D: -glucose (MeSAG) and methylsalicylate. Studies on the intracellular localization of the metabolites revealed that all of the SAG associated with tobacco protoplasts was localized in the vacuole. However, the majority of the MeSAG was located outside the vacuole. The tobacco cells contained an SA inducible SA glucosyltransferase (SAGT) enzyme that formed SAG. The SAGT enzyme was not associated with the vacuole and appeared to be a cytoplasmic enzyme. The vacuolar transport of SAG was characterized by measuring the uptake of [14C]SAG into tonoplast vesicles isolated from tobacco cell cultures. SAG uptake was stimulated eightfold by the addition of MgATP. The ATP-dependent uptake of SAG was inhibited by bafilomycin A1 (a specific inhibitor of the vacuolar H(+)-ATPase) and dissipation of the transtonoplast H(+)-electrochemical gradient. Vanadate was not an inhibitor of SAG uptake. Several beta-glucose conjugates were strong inhibitors of SAG uptake, whereas glutathione and glucuronide conjugates were only marginally inhibitory. The SAG uptake exhibited Michaelis-Menten type saturation kinetics with a K(m) and V(max) value of 11 microM and 205 pmol min-1 mg-1, respectively, for SAG. Based on the transport characteristics it appears as if the vacuolar uptake of SAG in tobacco cells occurs through an H(+)-antiport-type mechanism.

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max[L.] cv Williams 82) cell suspension cultures. [7–14C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–14C]SA was taken up rapidly from the culture media and converted primarily to SA 2‐O‐β‐d‐glucose (SAG). Lower levels of glucosylated 2,5‐dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2‐O‐β‐d‐glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP‐glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 mM SA. The SAGT activity in these cells was 88‐fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.