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Author: search.filters.author.Wany, AakankshaSubject: search.filters.subject.Nitrate

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    Methods for measuring nitrate reductase, nitrite levels, and nitric oxide from plant tissues
    (Springer Nature Publishing AG, 2020) Wany, Aakanksha; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
    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.
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    An overview of important enzymes involved in nitrogen assimilation of plants
    (Springer Nature Publishing AG, 2020) Kishorekumar, Reddy; Bulle, Mallesham; Wany, Aakanksha; Gupta, Kapuganti Jagadis
    Nitrogen (N) is a macro-nutrient that is essential for growth development and resistance against biotic and abiotic stresses of plants. Nitrogen is a constituent of amino acids, proteins, nucleic acids, chlorophyll, and various primary and secondary metabolites. The atmosphere contains huge amounts of nitrogen but it cannot be taken up directly by plants. Plants can take up nitrogen in the form of nitrate, ammonium, urea, nitrite, or a combination of all these forms. In addition, in various leguminous rhizobia, bacteria can convert atmospheric nitrogen to ammonia and supply it to the plants. The form of nitrogen nutrition is also important in plant growth and resistance against pathogens. Nitrogen content has an important function in crop yield. Nitrogen deficiency can cause reduced root growth, change in root architecture, reduced plant biomass, and reduced photosynthesis. Hence, understanding the function and regulation of N metabolism is important. Several enzymes and intermediates are involved in nitrogen assimilation. Here we provide an overview of the important enzymes such as nitrate reductase, nitrite reductase, glutamine synthase, GOGAT, glutamate dehydrogenase, and alanine aminotransferase that are involved in nitrogen metabolism.
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    Measurement of nitrate reductase activity in tomato (Solanum lycopersicum L.) leaves under different conditions
    (Springer Nature Publishing AG, 2020) Bulle, Mallesham; Kishorekumar, Reddy; Pathak, Pradeep K.; Wany, Aakanksha; Gupta, Kapuganti Jagadis
    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 1.6.6.1) 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.