Quantification of Symplasmic Phloem Loading Capacity with Live-Cell Microscopy

Abstract

Sugars produced by photosynthesis in leaves get transported to other organs in the phloem vascular tissue. Three general mechanisms have been proposed for the loading of sugars into the phloem. These differ in the involvement of active transport across the phloem cell’s membrane and their capacity for passive intercellular transport through plasmodesmata. This capacity for diffusion from the mesophyll into the phloem cells can be quantified by live-cell microscopy. Instead of sugar molecules, the movement of fluorescent tracers of similar size can be observed. In this chapter, a simple method is described that allows quantification of plasmodesmata-mediated intercellular diffusion across the mesophyll-bundle sheath interface and the bundle sheath-phloem cell interfaces. The fluorescent tracer carboxyfluorescein is loaded into intact leaves and its diffusion monitored with confocal microscopy after photobleaching of a bundle sheath cell.