title: Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.
abstract: The uptake of radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) by protoplasts isolated from leaves of Brassica napus was detected using silicone oil filtration technique. The uptake was pH-dependent with higher uptake rates at acidic pH. Imposition of a pH gradient (internal alkaline) across the plasma membrane resulted in a rapid uptake of p-OHBG, which was inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone, indicating that the uptake is dependent on a proton motive force. Dissipation of the internal positive membrane potential generated a small influx as compared with that seen for pH gradient (DeltapH). Kinetic studies demonstrated the presence of two uptake systems, a saturable and a linear component. The saturable kinetics indicated carrier-mediated translocation with a K(m) of 1.0 mm and a V(max) of 28.7 nmol/microl/h. The linear component had very low substrate affinity. The carrier-mediated transport had a temperature coefficient (Q(10)) of 1.8 +/- 0.2 in the temperature range from 4-30 degrees C. The uptake was against a concentration gradient and was sensitive to protonophores, uncouplers, H(+)-ATPase inhibitors, and the sulfhydryl group modifier p-chloromercuriphenylsulfonic acid. The carrier-mediated uptake system had high specificity for glucosinolates because glucosinolate degradation products, amino acids, sugars, or glutathione conjugates did not compete for p-OHBG uptake. Glucosinolates with different side chains were equally good competitors of p-OHBG uptake, which indicates that the uptake system has low specificity for the glucosinolate side chains. Our data provide the first evidence of an active transport of glucosinolates by a proton-coupled symporter in the plasma membrane of rape leaves.
authors.affiliation: Plant Biochemistry Laboratory, Department of Plant Biology, and Center for Molecular Plant Physiology (PlaCe), The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: B A
authors.initials: BA
authors.lastname: Halkier
mesh_terms: D001937:Brassica / Q000378:metabolism* / Q000648:ultrastructure; D005961:Glucosinolates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D018515:Plant Leaves / Q000378:metabolism / Q000648:ultrastructure; D011523:Protoplasts / Q000378:metabolism* / Q000502:physiology; D013696:Temperature
chemical_list: D005961:Glucosinolates