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    Review: Nutrient-nutrient interactions governing underground plant adaptation strategies in a heterogeneous environment
    (Elsevier B.V., 2024) Singh, Kratika; Gupta, Shreya; Singh, Amar Pal
    Plant growth relies on the mineral nutrients present in the rhizosphere. The distribution of nutrients in soils varies depending on their mobility and capacity to bind with soil particles. Consequently, plants often encounter either low or high levels of nutrients in the rhizosphere. Plant roots are the essential organs that sense changes in soil mineral content, leading to the activation of signaling pathways associated with the adjustment of plant architecture and metabolic responses. During differential availability of minerals in the rhizosphere, plants trigger adaptation strategies such as cellular remobilization of minerals, secretion of organic molecules, and the attenuation or enhancement of root growth to balance nutrient uptake. The interdependency, availability, and uptake of minerals, such as phosphorus (P), iron (Fe), zinc (Zn), potassium (K), nitrogen (N) forms, nitrate (NO3-), and ammonium (NH4+), modulate the root architecture and metabolic functioning of plants. Here, we summarized the interactions of major nutrients (N, P, K, Fe, Zn) in shaping root architecture, physiological responses, genetic components involved, and address the current challenges associated with nutrient-nutrient interactions. Furthermore, we discuss the major gaps and opportunities in the field for developing plants with improved nutrient uptake and use efficiency for sustainable agriculture.
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    Differential response of rice genotypes to nitrogen availability is associated with the altered nitrogen metabolism and ionomic balance
    (Elsevier B.V., 2022) Kumari, Priyanka; Devi, Loitongbam Lorinda; Kumar, Amresh; Pandey, Ashutosh; Sinha, Subodh Kumar; Singh, Amar Pal
    Nitrogen (N) uptake and its assimilation are crucial steps for plant growth and productivity. Plant's N balance largely depends on nitrate (NO3-) and ammonium (NH4+) forms present in the rhizosphere. Due to the fluctuating and heterogeneous availability of these N forms in the soils, plants encounter low to N deficiency. In contrast to low nitrogen, high N in the form of ammonium (NH4+) severely hampers plant development and causes NH4+ toxicity. In this study, we assessed eleven rice genotypes under sufficient (SN) and low N (LN) conditions. From the analysis, we identified a rice genotype, PB1, which is hypersensitive to SN and showed reduced root and shoot growth. In contrast to the SN condition, PB1 showed improved growth performance under the LN condition. Our data show that compromised growth of PB1 under SN condition is associated with increased activity of N responsive genes such as OsAMT1.1, OsAMT2.3, OsAMT3.1 and OsAMT3.2, OsNRT1.1A and OsNRT1.1B. Strikingly, LN treatment improved the root and shoot biomass with a concomitant increase in levels of NO3- and NH4+ transporter genes along with an increase in shoot: root NO3- ratio. Additionally, we show that increased levels of N in PB1 under SN condition are associated with the enhanced activity of the GS-GOGAT pathway. Further, our ionomic analysis highlighted the role of N-defined Fe accumulation which is partially associated with the N toxicity. Taken together, our study led to identifying a rice genotype (Oryza sativa L.) which is associated with enhanced N levels and assimilation and could be used for raising N use efficient rice varieties using breeding approaches.
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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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    Using different forms of nitrogen to study hypersensitive response elicited by avirulent Pseudomonas syringae
    (Springer Nature Publishing AG, 2020) Singh, Namrata; Singh, Pooja; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
    Nitrate, ammonium, or a combination of both is the form of N available for nitrogen assimilation from soil by the plants. Nitrogen is an important and integral part of amino acids, nucleotides, and defense molecules. Hence it is very important to study the role of nitrate and ammonium nutrition in plant defense via hypersensitive response (HR). Shifting plants from ammonium nitrate Hoagland solution to nitrate Hoagland nutrition slightly enhances root length and leaf area. HR phenotype is different in nitrate and ammonium grown plants when challenged with avirulent Pseudomonas syringae DC3000 avrRpm1. HR is also associated with increased production of reactive oxygen species (ROS) and nitric oxide (NO). Hence to understand HR development it is essential to measure HR lesions, cell death, ROS, NO, and bacterial growth. Here we provide a stepwise protocol of various parameters to study HR in Arabidopsis in response to nitrate and ammonium nutrition.
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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.
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    Using foldscope to monitor superoxide production and cell death during pathogen infection in arabidopsis under different nitrogen regimes
    (Springer Nature Publishing AG, 2020) Arora, Reena; Singh, Pooja; Kumari, Aprajita; Pathak, Pradeep Kumar; Gupta, Kapuganti Jagadis
    Nitrogen nutrition plays a role in plant growth development and resistance against biotic and abiotic stress. During pathogen infection various signal molecules such as reactive oxygen species, calcium, reactive nitrogen species, salicylic acid, and ethylene plays an important role. The form of nitrogen nutrition such as nitrate or ammonium plays a role in production of these molecules. Under nitrate nutrition NO is predominant. The produced NO plays a role in reacting with superoxide to generate peroxynitrite to induce cell death during hypersensitive response elicited by avirulent pathogens. Excess of ROS is also detrimental to plants and NO plays a role in regulating ROS. Hence it is important to observe superoxide production during infection. By using an avirulent Pseudomonas syringae and Arabidopsis differential N nutrition we show superoxide production in leaves using a paper microscope called Foldscope, which can be applied as a simple microscope to observe objects. The data also compared with root system infected with pathogenic Fusarium oxysporum. Taken together here we show that Foldscope is a cost-effective and powerful technique to visualize superoxide and cell death in plants during infection.