Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean (Glycine max [L.] cv Williams 82) cell suspension cultures. [7-C-14]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-C-14]SA was taken up rapidly from the culture media and converted primarily to SA 2-O-beta-d-glucose (SAG). Lower levels of glucosylated 2,5-dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2-O-beta-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.