A simple method for locating the start of symplastic water flow (flumes) in leaves

The identification of sites in leaves where transpiration water crosses cell membranes and enters the symplast has previously been made using freeze-substitution to locate concentrations of dye [e.g. sulphorhodamine (SR)] moving with the transpiration stream, and left outside the membranes where the water passes through. These concentrations were called sumps, and the sites of entry to the symplast were called flumes. A simple method of locating sumps, and therefore flumes, is described. Fresh leaves, fed SR solution through their cut petioles for pulse periods of 0.5 h or more, followed or not by a chase of water, were sectioned by hand under paraffin oil, and the sections mounted in the same fluid. Observation of the sections by simple bright-field microscopy revealed sumps of SR at the same sites, and of the same crystalline nature as found in the freeze-substituted preparations. The saving in preparation time is of the order of > 100-fold, at the sacrifice of resolution (5-10 μm compared with 0.2 μm). A limited survey of grass, sedge and dicotyledon leaves by this method confirmed in all essentials the results found by freeze-substitution, and in addition, revealed flumes at the fusoid cells on the flanks of the veins of bamboo leaves, and at the same position next to the water tissue of Cyperus leaves. The rate of accumulation of crystalline SR in the sumps inside tracheary elements suggests that the concentration of this non-permeating solute in the xylem sap increased by about 1000-fold in the finest veins during 1-2 h of transpiration in the dye solution.