Phloem as capacitor: radial transfer of water into xylem of tree stems occurs via symplastic transport in ray parenchyma

Abstract

The transfer of water from phloem into xylem is thought to mitigate increasing hydraulic tension in the vascular system of trees during the diel cycle of transpiration. Although a putative plant function, to date there is no direct evidence of such water transfer or the contributing pathways. Here, we trace the radial flow of water from the phloem into the xylem and investigate its diel variation. Introducing a fluorescent dye (0.1% [w/w] fluorescein) into the phloem water of the tree species Eucalyptus saligna allowed localization of the dye in phloem and xylem tissues using confocal laser scanning microscopy. Our results show that the majority of water transferred between the two tissues is facilitated via the symplast of horizontal ray parenchyma cells. The method also permitted assessment of the radial transfer of water during the diel cycle, where changes in water potential gradients between phloem and xylem determine the extent and direction of radial transfer. When injected during the morning, when xylem water potential rapidly declined, fluorescein was translocated, on average, farther into mature xylem (447 ± 188 µm) compared with nighttime, when xylem water potential was close to zero (155 ± 42 µm). These findings provide empirical evidence to support theoretical predictions of the role of phloem-xylem water transfer in the hydraulic functioning of plants. This method enables investigation of the role of phloem tissue as a dynamic capacitor for water storage and transfer and its contribution toward the maintenance of the functional integrity of xylem in trees.

Figure 1.

Micrographs showing anatomical structures in woody tissues of E. saligna. A, Intact free-hand transverse section showing phloem (right), cambium (c), and mature xylem tissue (left), including xylem parenchyma (xp) and vessels (v). Intact wood rays (r) traverse from phloem through the cambium into the xylem. Bar = 250 µm. B, Electron micrograph showing the transverse view of phloem, where rays extend through phloem parenchyma (pp) and sieve element/companion cell complexes (se/cc). Numerous calcium oxalate crystals are visible. Bar = 100 µm. C, Detailed transverse view of xylem vessels bordered by uniseriate rays surrounded by parenchymal tissue. Bar = 100 µm. D, Electron micrograph (radial plane) depicting vertical wood fibers (f) and the broad bands of nonvestured contact pits (cp) connecting the vessel with ray cells. Bar = 100 µm. Additional micrographs of wood anatomy are provided in Supplemental Figure S1.

Figure 2.

Staining characteristics (transverse view) several hours after injection of fluorescent dyes into phloem tissue of E. saligna and emission spectra of tissues and dyes. Arrows indicate the direction of water flow from phloem into xylem tissue. A, Injection wound (iw) of the needle used to introduce fluorescein in phloem tissue and migration of the dye in ray parenchyma. B, Transition of fluorescein from phloem toward xylem tissue. C, Fluorescein passing cambial tissue, entering mature xylem, and staining vessels. D, Injection wound where eosin Y was introduced and spread of the dye into phloem parenchymal tissue. E, Injection wound where rhodamin B was introduced, showing that the dye remained mostly stationary. F, Relative fluorescence emission spectra of woody tissues (phloem parenchyma and sapwood [peak 1]; autofluorescence), fluorescein (peak 2), eosin Y (peak 3), rhodamin B (peak 4), and chlorophyll (peak 5; autofluorescence). Micrographs are composite images of bandwidths scanned for woody tissue, fluorescent dye, and chlorophyll. Individual images of all channels are provided in Supplemental Figures S4 to S6. Bars = 200 µm.

Figure 3.

Environmental and physiological conditions during a diel time course (March 5, 2014). A, PAR and VPD. B, Diurnal course of ψ_L collected from the upper canopy of three E. saligna trees (n = 9 leaves per time point; error bars depict sd). C, Differential of the movement in phloem (black line) and xylem tissue (blue line) in basipetal stem sections of E. saligna (n = 3 trees; shaded areas represent sd) zeroed according to measurements recorded at time 0. Numbers and arrows indicate the phase of rapid shrinkage of phloem thickness during morning hours (peak 1) and growth during the night (peak 2); the gray dotted line marks the point where phloem rehydration is complete and wood growth occurs (for details, see text).

Figure 4.

Contrasting day versus nighttime progression of fluorescein (transverse view) in ray parenchyma of E. saligna. Dashed lines indicate borders between tissue types, where p = phloem, c = cambial zone and juvenile xylem, and x = mature xylem. Yellow bars show the maximum distance that fluorescein had progressed. A, Injection of the dye at 8 am, with tissue harvest at 12 noon. B, Injection of the dye at 7 pm, with tissue harvest prior to 7 am the following morning. Bars = 200 µm.