Methods for Measuring Nitrate Reductase, Nitrite Levels, and Nitric Oxide from Plant Tissues

Nitrogen (N) is one of the most important nutrients which exist in both inorganic and organic forms. Plants assimilate inorganic form of N [nitrate (NO₃^-), nitrite (NO₂^-) or ammonium (NH₄⁺)] and incorporate into amino acids. The metabolism of N involves a series of events such as sensing, uptake, and assimilation. The initial stage is sensing, triggered by nitrate or ammonium signals initiating signal transduction processes in N metabolism. The assimilation pathway initiates with NO₃^-/NH₄⁺ transport to roots via specific high and low affinity (HATs and LATs) nitrate transporters or directly via ammonium transporters (AMTs). In cytosol the NO₃^- is reduced to NO₂^- by cytosolic nitrate reductase (NR) and the produced NO₂^- is further reduced to NH₄⁺ by nitrite reductase (NiR) in plastids. NR has capability to reduce NO₂^- to nitric oxide (NO) under specific conditions such as hypoxia, low pH, and pathogen infection. The produced NO acts as a signal for wide range of processes such as plant growth development and stress. Here, we provide methods to measure NR activity, NO₂^- levels, and NO production in plant tissues.