Blockage of apoplastic bypass-flow of water in rice roots by insoluble salt precipitates analogous to a Pfeffer cell

Precipitates of insoluble inorganic salts were used to clog apoplastic pores in cell walls of the outer part of rice roots (OPR) in two rice cultivars (lowland cv. IR64 and upland cv. Azucena). Aerenchyma of two different root zones (20–50 and 50–100 mm from the apex) was perfused with 1 m m potassium ferrocyanide (K₄[Fe(CN)₆]) while the whole root segments were bathed in 0.5 m m copper sulphate (CuSO₄) medium. In another experiment, salts were applied on opposite sides of the OPR. The copper-ferrocyanide precipitation technique resembles the famous osmotic experiments of the German botanist Wilhelm Pfeffer, in which he used them with clay diaphragms. Precipitates were observed on the side where ferrocyanide was applied, suggesting that Cu²⁺ and SO₄^2– were passing the barrier including the Casparian bands of the exodermis much faster than ferrocyanide. There was a patchiness in the formation of precipitates, correlated with the maturation of the exodermis. The intensity of copper ferrocyanide staining decreased along developing rice roots. No precipitates were observed in mature parts beyond 70–80 mm from the root apex, except for sites around the emergence of secondary roots, which were fairly leaky to both water and ions. Blockage of the apoplastic pores with precipitates caused a three- to four-fold reduction of hydraulic conductivity of the OPR (Lp_OPR). The reflection coefficient of the OPR (σ_sOPR) increased in response to the blockage with precipitates. The osmotic versus diffusive water permeability ratios of the OPR (P_fOPR/P_dOPR) were around 600 for immature and 1200 for mature root segments. Treatment significantly affected the bulk rather than the diffusive water flow and caused a three- to five-fold reduction of the P_fOPR/P_dOPR ratios. Results indicated that despite the existence of an exodermis with Casparian bands, most of the water moved around cells rather than using the cell-to-cell passage.     

A new precipitation technique provides evidence for the permeability of Casparian bands to ions in young roots of corn (Zea mays L.) and rice (Oryza sativa L.)

Using an insoluble inorganic salt precipitation technique, the permeability of cell walls and especially of endodermal Casparian bands (CBs) for ions was tested in young roots of corn (Zea mays) and rice (Oryza sativa). The test was based on suction of either 100 µm CuSO₄ or 200 µm K₄[Fe(CN)₆] into the root from its medium using a pump (excised roots) or transpirational stream (intact seedlings), and subsequent perfusion of xylem of those root segments with the opposite salt component, which resulted in precipitation of insoluble brown crystals of copper ferrocyanide. Under suction, Cu²⁺ could cross the endodermis apoplastically in both plant species (although at low rates) developing brown salt precipitates in cell walls of early metaxylem and in the region between CBs and functioning metaxylem vessels. Hence, at least Cu²⁺ did cross the endodermis dragged along with the water. The results suggested that CBs were not perfect barriers to apoplastic ion fluxes. In contrast, ferrocyanide ions failed to cross the mature endodermis of both corn and rice at detectable amounts. The concentration limit of apoplastic copper was 0.8 µm at a perfusion with 200 µm K₄[Fe(CN)₆]. Asymmetric development of precipitates suggested that the cation, Cu²⁺, moved faster than the anion, [Fe(CN)₆]^4–, through cell walls including CBs. Using Chara cell wall preparations (‘ghosts’) as a model system, it was observed that, different from Cu²⁺, ferrocyanide ions remained inside wall-tubes suggesting a substantially lower permeability of the latter which agreed with the finding of an asymmetric development of precipitates. In both corn and rice roots, there was a significant apoplastic flux of ions in regions where laterals penetrated the endodermis. Overall, the results show that the permeability of CBs to ions is not zero. CBs do not represent a perfect barrier for ions, as is usually thought. The permeability of CBs may vary depending on growth conditions which are known to affect the intensity of formation of bands.