Root Ecophysiology
Visualization of glutathione conjugation
Glutathione conjugation can be visualized in living cells using substrates that emit fluorescence after conjugation. Monochlorobimane (MCB) is such a substrate.
The development of fluorescence in onion epidermal protoplast preparations in vivo was followed microscopically. Fluorescence development occurred rapidly within a few minutes after incubation with MCB. When fluorescence development was monitored in nuclear, vacuolar and cytosolic positions, cytosolic and vacuolar fluorescence showed the same increase, which is most probably due to an overshining of cytosolic fluorescence into the vacuole. The fluorescence is thought to be attributable to a transport and preferential distribution of the conjugate into the vacuole of the cells, as could be shown with isolated vacuoles.
from Schröder and Stampfl 1999
Visualization of MCB dependent glutathione conjugation in barley
The model xenobiotic, monobromobimane (MCB), is rapidly taken up, conjugated to GSH in the root tip and transported basipetally in the xylem of barley roots. Note the strong fluorescence in the meristem.
Distribution of GSB and GSH content was imaged in different cell compartents in barley roots 30 min after treatment with MCB (50µM). The image shows regions of bright small cells in the meristematic tissue of the root.
The fluorescent bimane conjugate distributes in epidermal cells, and also in the vacuoles of root hairs.
from Schröder et al. 2002, Presented as a poster at the 2002 IUPAC Conference, Basel.
Visualization of microsomal glutathione conjugation
The formation of a fluorescence glutathione conjugate was followed microscopically in the microsomal leaf extract of P. erosus (EC 550). Fig. 2 shows the time course of development of GSH-MCB fluorescence conjugate. To our knowledge this image provides the first fluorescence description of microsomal GST catalysed reaction in a legume. Fluorescence development begins immediately after incubation with just a tiny spot on single microsomes intensifying up to 16 min. At 20 min a shining fluorescence appeared and brightly expressed after 22 min. The increased appearance of this fluorescence conjugate provides visual evidence of the catalytic conversion of monochlorobimane and confirms production of its nonphytotoxic glutathione conjugate.
