Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treat ment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addi tion of [C-14]-labeled scopoletin showed that scopoletin was taken up by 2, 4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Exp eriments using several inhibitors showed that this uptake was energy-depend ent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydr oxycoumarins, such as scopoletin, umbelliferone and esculetin. To further i nvestigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, th erefore, is likely to be located in the cytosol. Therefore, we can state th at 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopo letin was converted into scopolin in the cytoplasm, and then transferred in to the vacuoles. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved .