Measurement of Nitrate Reductase Activity in Tomato (Solanum lycopersicum L.) Leaves Under Different Conditions

  • Mallesham Bulle
  • Reddy Kishorekumar
  • Pradeep K. Pathak
  • Aakanksha Wany
  • Kapuganti Jagadis Gupta
Part of the Methods in Molecular Biology book series (MIMB, volume 2057)


Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO3) or ammonium (NH4+) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH4+ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO3 is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO2) by nitrate reductase (NR) (EC which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO3. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO3 to NO2 under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.

Key words

Nitrate reductase Hypoxia Light Nitrate Nitrite Nitric oxide Ammonium 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Mallesham Bulle
    • 1
  • Reddy Kishorekumar
    • 1
  • Pradeep K. Pathak
    • 1
  • Aakanksha Wany
    • 1
  • Kapuganti Jagadis Gupta
    • 1
  1. 1.National Institute of Plant Genome ResearchNew DelhiIndia

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