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

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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.
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    Localization of nitric oxide in wheat roots by DAF fluorescence
    (Springer, 2016) Wany, Aakanksha; Gupta, Kapuganti Jagadis
    Nitric oxide is a free radical signal molecule. Various methods are available for measurement of NO. Out of all methods, fluorescent probes to localize NO is very widely used method. Diaminofluorescein in diacetate form (DAF-2DA) is most widely probe for NO measurement. This method is based on application of 4,5-diaminofluorescein diacetate (DAF-2DA) which is actively diffused into cells, once taken up by cells cytoplasmic esterases cleave the acetate groups to generate 4,5-diaminofluorescein; DAF-2. The generated DAF-2 can readily react with N2O3, which is an oxidation product of NO to generate the highly fluorescent DAF-2T (triazolofluorescein). There are various advantages and disadvantages associated with this method, but to its advantage in diffusion closely to NO producing sites, it is widely used for localization studies. Here, we describe method to make sections of the roots and localization of NO in roots subjected to hypoxic stress.