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 (ΔpH). 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/μl/h. The linear component had very low substrate affinity. The carrier-mediated transport bad a temperature coefficient (Q10) of 1.8 ± 0.2 in the temperature range from 4-30 °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.