Measurement of Net Fluxes of Ammonium and Nitrate at the Surface of Barley Roots Using Ion-Selective Microelectrodes

Abstract

Neutral carrier-based liquid membrane ion-selective microelectrodes for NH4+ and N03-were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium-and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH4+ and N03-were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH4+ into the cold-adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH4+ and N03-were present. Similarly, net flux of N03-into the warm-adapted variety was not affected when NH4+ was also present in solution. The high temporal and spatial resolution afforded by ammonium-and nitrateselective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions.

Investigation of the Apparent Induction of Nitrate Uptake in Barley (Hordeum vulgare L.) Using NO3--Selective Microelectrodes (Modulation of Coarse Regulation of NO3- Uptake by Exogenous Application of Downstream Metabolites in the NO3- Assimilatory Pathway)

“…The fabrication and characteristics of N03--selective microelectrodes, as well as the microelectrode system used to determine net ionic fluxes at the surface of intact roots, have been described previously (Henriksen et al, 1990(Henriksen et al, , 1992. Briefly, micropipets were pulled from 2.0-mm 0.d.…”

“…Plants to be used for net flux experiments were transfemed into the experimental chamber (Henriksen et al, 1990, figure 1; Henriksen et al, 1992, figure 2) at least 12 h before the start of an experiment. Nutrient solution from a 3-L reservoir was circulated through the chamber (130 mL volume) at 50 mL min-I.…”

“…Following the pretreatment period, the plants were exposed to NOs-, and net NO3-fluxes were measured at a single axial location using N03--selective microelectrodes in the unstirred layer of solution immediately extemal to the root surface (Henriksen et al, 1990(Henriksen et al, ,1992. Using this technique, we could continuously monitor the induction phenomenon over a small surface element of a single intact root, thus avoiding the averaging that occurs when whole root systems and populations of plants are used to investigate the induction of NOs-uptake.…”

Construction, Theory, and Practical Considerations for using Self-referencing of Ca2+-Selective Microelectrodes for Monitoring Extracellular Ca2+ Gradients

“…the quantity of substance (Q) moving per unit time (Henriksen et al, 1992). Flow is the same for all concentric surfaces surrounding the source surface.…”

“…A caveat of this approach is the assumption that the flow is equal at all points around the cylinder and along the shaft of the cylinder. An alternative is to calculate flux per unit length by dividing by r  2 (Henriksen et al, 1992).…”