Poster: Membrane transport
Abs #
165: Formation and vacuolar transport of salicylic acid glucose conjugates in tobacco cell suspension cultures
The metabolism and vacuolar transport of salicylic acid (SA) was investigated in tobacco cell suspension cultures. [14C]SA was added to tobacco cells and the metabolites were extracted 24 h after treatment. Nearly 90 % of the radioactivity was recovered in the media (16%), the cell debris (21%), or the cell extract (53%). Three main metabolites of SA were identified in the cell extract. SA-2-O-β-D-glucose (SAG) and methylsalicylate-2-O-β-D-glucose (MeSAG) were the major metabolites formed and methylsalicylate (MeSA) was a minor metabolite. Examination of the intracellular localization of SAG and MeSAG revealed that most of the SAG was found in the vacuoles; whereas, the level of MeSAG in vacuoles was consistently lower than the level of SAG. The vacuolar transport of SA, SAG, and MeSAG was measured using tobacco tonoplast vesicles. The uptake of SAG into tonoplast vesicles in the presence of ATP was 9-fold greater than the uptake in the absence of ATP. The ATP-dependent uptake was saturable with a Km of 11 μM and was strongly inhibited by a variety of phenolic β-glucosides but not by phenolic α-glucosides. The ATP stimulated uptake of SAG was inhibited by gramicidin (cation-specific ionophore; 60% inhibition); and bafilomycin A1 (vacuolar H+-ATPase inhibitor; 80 % inhibition), but not by vanadate (ABC transporter inhibitor). Therefore, vacuolar uptake of SAG in tobacco may be due to an H+-antiport-type mechanism. Uptake of SA and MeSAG by tobacco tonoplast vesicles was only slightly stimulated in the presence of ATP and was 2.5-fold and 2.7-fold, respectively, less than the ATP-dependent uptake of SAG. The low level of MeSAG uptake into tobacco tonoplast vesicles is consistent with the low levels of MeSAG found in intact tobacco vacuoles.
