Analysis of 13NH4+ Efflux in Spruce Roots (A Test Case for Phase Identification in Compartmental Analysis)

Abstract

111. Based on these findings and the assumption of an in-series arrangement of root cell compartments, it was concluded that phase 111 corresponded to the cytoplasm, phase II corresponded to the Donnan free space, and phase I corresponded to a film of solution adhering to the root surface.Efflux analysis is widely used to determine unidirectional ion fluxes, kinetic exchange constants of subcellular compartments, and ionic concentrations within compartments. In plants, compartmental analyses have been undertaken for a variety of ions, including Na+, K+, Mg2+, Ca2+, NH,+, NO,-, Pi, SO,'-, C1-, and Br-(for refs., see Wang, 1994). The majority of efflux studies have been limited to nonmetabolized ions and were performed usually on excised tissues or suspension-culture systems, mainly because of the (presumed) absence of complicating factors such as metabolism and long-distance transport to the shoot (Pitman, 1963; Cram, 1968;Poole, 1971aPoole, , 1971b Macklon, 1975a, 197513; Sim, 1976, 1981;Pfriiner and Bentrup, 1978;Macklon et al., 1990). For use with intact plant material, a detailed treatise on parameter extraction was presented by Jeschke and Jambor (1981) and Jeschke (1982). More recently, compartmental analysis has also been applied to metabolized ions, including S0,2p (Thoiron et al., 1981;Cram, 1983; Bell et al., 1994), Pi (Lefebvre and Clarkson, 1984;Macklon and Sim, 1992), NO,-(Presland and McNaughton, 1984 1986; Macklon et al., 1990;Siddiqi et al., 1991;Devienne et al., 1994;Kronzucker et al., 1995a Kronzucker et al., , 1995b, and NH,+ (Presland and McNaughton, 1986;Cooper et al., 1989;Macklon et al., 1990;Wang et al., 1993a;Kronzucker et al., 1995~). Despite this widespread use of the technique, workers have typically neglected to conduct physiological tests to verify the subcellular identities of the phases revealed in efflux data. As a consequence of this omission, the assignment of particular kinetically defined phases to their corresponding subcellular compartments has not always been unequivocal (Macklon et al., 1990). Only in studies by Cram (1968), Lee and Clarkson (1986), and Siddiqi et al.(19911, as well as in a previous study of NO,-exchange in spruce (Kronzucker et al., 1995a), was the assignment of compartments substantiated. Usually phase assignment has been based on the assumption of an in-series arrangement of cell compartments, i.e. cell wall, cytoplasm, and vacuole (Pitman, 1963; Cram, 1968Cram, , 1975. Thus, the first (rapidly exchanging) phase has been assumed to represent the cell wall and the last (slowest exchanging) phase has been assumed to represent the vacuole. However, the derivation of flux components as well as of pool sizes from efflux data is valid only if subcellular compartments are assigned correctly to their corresponding efflux phases.We have used a combination of strategies to analyze the efflux reported in the present study to distinguish between membrane-bound and metabolically dependent (intracellular) compartments and those that are nonmembrane bound and app...

Inhibition of Nitrate Uptake by Ammonium in Barley. Analysis of Component Fluxes1

“…We chose the duration of the loading period on the basis of the half-lives of exchange for the cytoplasmic compartment for NO 3 Ϫ in barley (compare below with Siddiqi et al, 1991). Therefore, 60 min of exposure to tracer ensured that cytoplasmic specific activity was Ն95% of that in the loading solution (Kronzucker et al, 1995e). After loading with 13 N, seedlings were transferred to efflux funnels (Siddiqi et al, 1991;Kronzucker et al, 1995b) and the roots were eluted with 20-mL aliquots of nonradioactive solution after varying time intervals.…”

Futile transmembrane NH 4 + cycling: A cellular hypothesis to explain ammonium toxicity in plants

“…Others have suggested that carbohydrate limitation may contribute to the toxicity syndrome, based on the finding that NH 4 ϩ per se is not translocated to the shoot in most plants (14), and, thus, all C skeletons for N assimilation must be provided in roots, causing local C deprivation (15). In some cases, external provision of ␣-ketoglutarate, a key carbon source for N assimilation, alleviated toxicity symptoms (4), but in other cases it failed to enhance NH 4 ϩ metabolism (16), suggesting that other factors may limit NH 4 ϩ assimilation. The hypothesis that NH 4 ϩ toxicity results from the uncoupling of photophosphorylation in chloroplasts (6) has long been shown to be incorrect, as even very large NH 4 ϩ concentrations do not affect this process in intact chloroplasts (17).…”

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

“…Another major limitation is that at times, it can be difficult to discriminate between fluxes that are occurring across membranes and those occurring extracellularly. Such distinctions call for rigorous phase testing 7,10,20 . 10 , but from extracellular spaces (c.f., Figure 3).…”

“…as a function of elution time. For steady-state conditions, perform linear regressions and calculations of fluxes, half-lives of exchange, and pool sizes (for details, see [6][7][8][9] ). ).…”

“…We should note from the outset that this article simply describes the steps necessary to perform each protocol. Where appropriate, brief explanations of calculations and theory are provided, but detailed expositions of each technique's background and theory can be found in several key articles on the subject 4,[6][7][8][9] . Importantly, these protocols are broadly transferable to flux analysis of other nutrients/toxicants (e.g., ) and to other plant species, albeit with a few caveats (see below).…”