Modification of the pressure-probe technique permits controlled intracellular microinjection of fluorescent probes

Abstract

ABSTRACT The pressure probe has been widely used to study the water relations of plant cells. Here we describe a simple modification of the pressure-probe technique that permits the controlled microinjection of fluorescent probes into plant cells while simultaneously measuring cell turgor pressure. Using the pressure probe, less than 1 nl of the membrane-impermeant fluorescent dye Lucifer Yellow CH was introduced into micropipettes and subsequently injected into leaf trichome cells of Nicotiana clevelandii. Disruption of cell contents could be minimized by raising the hydrostatic pressure in the probe prior to impalement to a value approaching the anticipated cell turgor pressure. Injections to the cytosol resulted in intercellular symplastic transport of the dye in both acropetal and basipetal directions. In contrast, no symplastic transport was observed following an injection of dye into the vacuole. As measured with the pressure probe, cell turgor pressures were in the range 0.18 to 0.36 MPa; the half-time for water exchange across the cell boundary was approximately 10 s. The potential of this technique for the study of turgor-pressure-dependent intercellular transport and the hydraulic conductivities of the tonoplast, plasmalemma and plasmodesmata is discussed.