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 (NH4+) as the sole nitrogen source. Recently, NH4+ toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying NH4+ 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 NH4+ 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 NH4+. We show that, at high external NH4+ concentration ([NH4+]o), barley root cells experience a breakdown in the regulation of NH4+ influx, leading to the accumulation of excessive amounts of NH4+ in the cytosol. Measurements of NH4+ efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for NH4+, reveal that, at elevated [NH4+]o, barley cells engage a high-capacity NH4+-efflux system that supports outward NH4+ 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 NH4+ at high [NH4+]o. Thus our study has characterized the primary events in NH4+ nutrition at the cellular level that may constitute the fundamental cause of NH4+ toxicity in plants.