Rapid induction of NH₄⁺ efflux in rice roots under high-ammonium stress and its association with varietal differences in ammonium tolerance and ammonium-utilization efficiency

Ammonium (NH₄⁺) efflux is a pivotal mechanism underlying root responses to NH₄⁺ toxicity, yet critical questions have remained unresolved, such as on the speed of onset and rectification of the NH₄⁺-efflux response to NH₄⁺ stress, its association with ammonium-use efficiency (AUE) and NH₄⁺ tolerance, and the identity of the transporter proteins or channel types mediating efflux. Here we survey the current state of the literature on the topic and combine the survey with new experiments in the leading crop species rice. We provide a pharmacological profile of NH₄⁺ fluxes under transient high-NH₄⁺ treatments in rice-root protoplasts and demonstrate that acute NH₄⁺ stress induces immediate and sustained NH₄⁺ efflux, likely mediated by aquaporins, ATP-Binding Cassette transporters, and potassium channels. We furthermore provide a screen of 99 cultivars that reveals a strong negative correlation (P < 0.01) between NH₄⁺ efflux and both root-NH₄⁺ tolerance and AUE, identifying efflux as a critical constraint on nitrogen-use efficiency. Probenecid-mediated inhibition of efflux is shown to boost NH₄⁺ uptake by 23.65% in high-efflux cultivars, underscoring the regulatory role of NH₄⁺ efflux. Our findings shine new light on plant adaptation to NH₄⁺ toxicity and establish a molecular framework for improving nitrogen-use-efficiency of crops through modulation of NH₄⁺-efflux pathways.