Measurement of Net NH 4 + Fluxes Using the Non-invasive Micro-Test Technology (NMT) System in Rice.

Abstract

Ammonium (NH 4 + ) is the primary inorganic nitrogen source for rice ( Oryza sativa L.). Substantial progress has been made in characterizing the functions of ammonium transporters (AMTs) in roots; however, the regulatory dynamics governing subcellular ammonium compartmentation after its entry into cells, particularly its vacuolar sequestration and efflux back to the external environment, remain poorly understood. This knowledge gap stems mainly from two factors: the difficulty of applying conventional detection methods at the organellar scale and interference caused by nonspecific ion adsorption to the cell wall of intact roots. To address these challenges, we present a detailed and reproducible protocol for real-time measurement of net NH 4 + fluxes in rice roots, root protoplasts, and isolated vacuoles using non-invasive micro-test technology (NMT). The protocol covers the preparation of protoplasts and vacuoles from rice roots, the configuration and calibration of the NMT system, and the step-by-step measurement of net NH 4 + fluxes at three distinct biological levels (intact roots, protoplasts, and vacuoles). By employing a unified sample preparation and measurement strategy, this protocol enables quantification of net uptake fluxes across the plasma membrane, characterization of net efflux dynamics under specific conditions, and indirect estimation of vacuolar sequestration capacity using the isolated vacuole system. Overall, this protocol provides a flexible and robust framework for studying NH 4 + homeostasis in plants and is readily adaptable to different crop species, treatment conditions, and experimental objectives. Owing to its modular design and compatibility with standard NMT equipment, it can be readily adopted by laboratories seeking to investigate nitrogen transport mechanisms in plants. Key features • Allows for testing of NH 4 + fluxes in roots, protoplasts, and vacuoles. • Applicable to plants grown under different culture systems, including Arabidopsis thaliana grown in dishes and rice grown in hydroponic systems. • Supports both long-term and transient stress treatments. • Real-time monitoring.

Plant phenotyping relevance

植物根・プロトプラスト・液胞のNH4+フラックスをリアルタイム定量するNMT測定プロトコルが研究の中心であり、植物の生理状態を取得する方法を詳細に開発・標準化している。

Code and data availability

This is a Bio-protocol methods article describing NMT measurement of NH4+ fluxes in rice. No public phenotype/trait datasets, plant images, author analysis code, or trained models are deposited; data acquisition uses proprietary imFluxes V3.0 software and analysis uses GraphPad Prism, with no availability statements or

No evidence-backed public reproduction asset is currently recorded.