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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.

The apoplastic tracer sulphorhodamine G (SR) was used as an indicator of the flumes, the sites where water left the apoplast and entered the symplast, in a selection of dicotyledon leaves. At these flumes the dye is deposited as crystals after a pulse of dye is fed to the transpiration stream, followed or not by a water chase. In contrast to wheat, the dicotyledons showed SR cystals inside the tracheary elements of the finest leaf veins. At short pulse times the crystals were in the stems of the branch-trees of the fine veins, but after longer pulses, had moved to the vein termini. The dye solution was moving very slowly in the tracheary elements as it approached the ends of the branch-trees, since the axial flow there is nearly balanced by radial leakage. These results are interpreted as evidence that most of the transpiration water enters the symplast in the vein sheaths of the fine veins, and that these veins are places where many of the natural solutes of the xylem sap will be enriched to quite high concentrations.

The fluorescent tracers sulphorhodamine G (SR) and pyrene trisulphonate (PTS) were used to explore the functions of cells and tissues within the pine needle, following their progress after feeding through the transpiration stream. The distributions of tracer in the needles were stabilized for fluorescence microscopy by rapid freezing and freeze-substitution, and anhydrous embedding and sectioning. After short pulse + chase times (up to 2 h), SR and PTS accumulated at higher-than-xylem concentrations in the transfusion tracheids on the flanks of the vascular strand, but did not pass out through the endodermis. The accumulations occurred locally where the transpiration water was separated from the tracers and passed into the symplast of the transfusion parenchyma and endodermis. After a 24 h water chase, SR had entered the symplast through the transfusion parenchyma, and spread through the endodermis and palisade. It is argued that this is evidence of active H+-ATPase systems lowering the external pH of the transfusion parenchyma, and characterizes these cells as scavenging cells similar to those found in the bundle sheath systems of legume leaves. The transport of SR through the endodermis and palisade is the first clear evidence that this tracer can also function as a symplastic tracer. The hypothesis that the transfusion parenchyma acts to scavenge solutes from the transpiration stream was tested by loading the stream with [14C]aspartate and examining the subsequent distribution of 14C by dry autoradiography. After a pulse + chase of (0.75 + 3) h, 14C was found concentrated in the transfusion parenchyma, and at even higher levels in the Strasburger cells. It is proposed that major functions of the transfusion tissue of gymnosperms are (a) the concentrating of solutes from the transpiration stream and (b) the retrieval from the stream of selected solutes that are returned to the phloem through the Strasburger cells, or forwarded through the endodermis to the palisade.

Following our publication of a new method of calculating rates of water uptake by roots from measurements of the rate of accumulation on the roots of a marker solute, this paper describes the sites of accumulation of the solute, which indicate the sites where the water entered the symplast. Sulphorhodamine G (SR) was supplied in aeroponic mist culture to large maize plants with fully developed root systems. Root samples were collected after 4 to 8 h of transpiration in the dye-mist from both axes and branches of the main roots, and from non-transpiring (detopped) controls, frozen rapidly, freeze-substituted, and embedded and sectioned by an anhydrous procedure that preserves the SR in place. Whole mounts and sections were examined by bright-field, polarizing and epifluorescence microscopy. Major accumulations of SR were all at the outer surface of the roots, on epidermal or root hair cell walls, or, in older roots where the epidermal cells were separating or dead, on the outer wall of the hypodermis. On some branch roots, though not on any main axes, the accumulations of SR were conspicuously aligned in the grooves over anticlinal cell walls of the epidermis. Non-transpiring plants showed very slight accumulations. Diffusion of SR into the cell wall apoplast was limited by the suberized lamellae and Casparian bands of the hypodermis, except in some branch roots, where SR diffused throughout the cortical cell walls. In parts of roots where the epidermis and hypodermis had been damaged, SR diffused through cell walls of the cortex from the wound site. These patterns of accumulation show that water enters the symplast of roots at the outermost cell membranes of the root, whether they are epidermal or hypodermal cells. Water enters roots with fully developed hypodermises at high rates. The role of the hypodermal suberization is to limit solute movement in the wall apoplast. A symplastic path for water throughout the cortex, endodermis and living cells of the stele is suggested.