Futile transmembrane NH4(+) cycling: a cellular hypothesis to explain ammonium toxicity in plants

Abstract

Most higher plants develop severe toxicity symptoms when grown on ammonium (NH(4)(+)) as the sole nitrogen source. Recently, NH(4)(+) toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying NH(4)(+) toxicity have been extensively sought, the primary events conferring it at the cellular level are not understood. Using a high-precision positron tracing technique, we here present a cell-physiological characterization of NH(4)(+) acquisition in two major cereals, barley (Hordeum vulgare), known to be susceptible to toxicity, and rice (Oryza sativa), known for its exceptional tolerance to even high levels of NH(4)(+). We show that, at high external NH(4)(+) concentration ([NH(4)(+)](o)), barley root cells experience a breakdown in the regulation of NH(4)(+) influx, leading to the accumulation of excessive amounts of NH(4)(+) in the cytosol. Measurements of NH(4)(+) efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for NH(4)(+), reveal that, at elevated [NH(4)(+)](o), barley cells engage a high-capacity NH(4)(+)-efflux system that supports outward NH(4)(+) fluxes against a sizable gradient. Ammonium efflux is shown to constitute as much as 80% of primary influx, resulting in a never-before-documented futile cycling of nitrogen across the plasma membrane of root cells. This futile cycling carries a high energetic cost (we record a 40% increase in root respiration) that is independent of N metabolism and is accompanied by a decline in growth. In rice, by contrast, a cellular defense strategy has evolved that is characterized by an energetically neutral, near-Nernstian, equilibration of NH(4)(+) at high [NH(4)(+)](o). Thus our study has characterized the primary events in NH(4)(+) nutrition at the cellular level that may constitute the fundamental cause of NH(4)(+) toxicity in plants.

Figure 1

Semilogarithmic plots of ¹³NH efflux from the cytosolic compartments of barley and rice roots. Plants were prelabeled under steady-state conditions, with NH provided externally at 10 mM. Plots have been corrected for specific activities of ¹³N tracer (2, 21), allowing a direct comparison of initial efflux rates by inspection of the y-intercepts of the regression lines.

Figure 2

Comparison of steady-state bidirectional plasma-membrane NH fluxes in barley and rice roots at 0.1 mM and 10 mM [NH]_o. Column height represents total influx from the external medium (Φ_oc), while the filled areas depict the portion of influx returned to the environment by efflux transport (Φ_co). The net flux (Φ_net) is the difference between these two fluxes. Vertical bars indicate standard errors of influx means.

Figure 3

Respiration rates of intact barley roots at 0.1 mM and 10 mM [NH]_o. In one experiment, 1 mM methionine sulfoximine (MSX) was applied to block NH metabolism (see text). Relative growth rates under the two conditions are shown in the Inset. Rice experienced no significant difference in respiration under the two NH regimes (see text).