Confocal laser scanning microscopy (CLSM) has had wide application in morph
ological studies and ion imaging in plants, but little impact so far on bio
chemical investigations. This position is likely to change as the range of
fluorescent probes increases. To illustrate the type of kinetic information
that can be obtained using CLSM in an intact, living system, an analysis h
as been made of the two-step detoxification of monochlorobimane (MCB) follo
wing conjugation to glutathione (GSH) by a glutathione S-transferase in the
cytoplasm and vacuolar sequestration of the fluorescent glutathione S-bima
ne (GSB) by a glutathione S-conjugate (GSX) pump, Fluorescence from the cyt
oplasm and vacuole of individual trichoblasts and atrichoblasts was measure
d from time-series of (x, y) optical sections in the elongation zone of Ara
bidopsis root tips. Intensity changes were calibrated and converted to amou
nts using compartment volumes, measured by stereological techniques. The da
ta were well described using pseudo-first-order kinetics for the conjugatio
n reaction and either Michaelis-Menten kinetics (Model I), or, as the GSX-p
ump was operating close to V-max a pseudo-zero-order reaction (Model II), f
or the GSX-pump, Analysis of 15 individual cells from two roots gave [GSH](
cyt) in the range 1.8-4 mM, GST activity was relatively constant on a cell
basis in one root, but increased markedly in the other, giving a net increa
se in conjugation activity as cells progressed through the elongation zone.
In contrast, GSX-pump activity increased in parallel with the increase in
cell size in both roots, effectively maintaining a constant transport activ
ity per unit root length or estimated vacuole surface area.