Browsing by Author "Wany, Aakanksha"

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Chemiluminescence detection of nitric oxide from roots, leaves, and root mitochondria
(Springer, 2016) Wany, Aakanksha; Gupta, Alok Kumar; Kumari, Aprajita; Gupta, Shika; Mishra, Sonal; Jaintu, Ritika; Pathak, Pradeep K.; Gupta, Kapuganti Jagadis
NO is a free radical with short half-life and high reactivity; due to its physiochemical properties it is very difficult to detect the concentrations precisely. Chemiluminescence is one of the robust methods to quantify NO. Detection of NO by this method is based on reaction of nitric oxide with ozone which leads to emission of light and amount of light is proportional to NO. By this method NO can be measured in the range of pico moles to nano moles range. Using direct chemiluminescence method, NO emitted into the gas stream can be detected whereas using indirect chemiluminescence oxidized forms of NO can also be detected. We detected NO using purified nitrate reductase, mitochondria, cell suspensions, and roots; detail measurement method is described here.
Current approaches to measure nitric oxide in plants
(Oxford University Press, 2019) Vishwakarma, Abhaypratap; Wany, Aakanksha; Pandey, Sonika; Bulle, Mallesham; Kumari, Aprajita; Kishorekumar, Reddy; Igamberdiev, Abir U.; Mur, Luis A.J.; Gupta, Kapuganti Jagadis
Nitric oxide (NO), is now established as an important signalling molecule in plants where they influence growth, development and responses to stress. Despite of extensive research, the most appropriate methods to measure and localise these signalling radicals are debated and still needs investigation. Many confounding factors such as presence of other reactive intermediates, scavenging enzymes and compartmentation, influence how accurately each can be measured. Further, these signalling radicals have short half-lives ranging from seconds to minutes based on the cellular redox condition. Hence, it is necessary to use the sensitive and specific methods in order to understand the contribution of each signalling molecule to various biological processes. In this review, we will provide current state knowledge on NO measurement in plant samples, via various methods. We will also discuss advantages, limitations and wider applications of each method.
Expression analysis of important genes involved in nitrogen metabolism under hypoxia
(Springer Nature Publishing AG, 2020) Bulle, Mallesham; Kishorekumar, Reddy; Wany, Aakanksha; Gupta, Kapuganti Jagadis
Hypoxia or anoxia condition can occurs during flooding or waterlogging and can cause intense damage to the plants. Since oxygen is important for active operation of electron transport chain in mitochondria to generate energy production (ATP) any drop in oxygen can cause an energy crisis during flooding/waterlogging. To cope with this energy crisis plants have developed various anatomical, physiological, and biochemical adaptations. Perception of signals and induction of genes are required for initiation of these adaptive responses. Various genes involved in nitrogen, carbon, and fermentative metabolism play a role in hypoxic tolerance. Regulation of genes involved in nitrogen metabolism also plays a role under hypoxia. Hence in this present chapter we describe the expression of nitrate reductase-1 (NIA1), nitrate reductase-2 (NIA2), and glutamine synthetase-1 (GLN-1) during hypoxia in Arabidopsis.
The functional role of nitric oxide in plant mitochondrial metabolism
(Elsevier B.V., 2016) Gupta, Alok Kumar; Kumari, Aprajita; Mishra, Sonal; Wany, Aakanksha; Gupta, Kapuganti Jagadis
In recent years, mitochondrial nitric oxide (NO) production has attracted increasing attention. Mitochondria generate NO using nitrite as a substrate. Cytochrome c oxidase and other components of the electron transport chain also contribute to NO generation. Accumulating evidence indicates that mitochondria are scavengers of NO. Furthermore, several genes encoding mitochondrial proteins, as well as mitochondrial proteins, are regulated by NO. In this chapter, we provided an overview of the mechanisms of NO generation and scavenging in mitochondria and of the NO-dependent regulation of proteins and genes encoding mitochondrial proteins. In addition, the functional roles of NO in mitochondrial metabolism, such as inhibition of aconitase, production of ATP and induction of alternative oxidase are presented.
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.
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.
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.
Nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia in Arabidopsis
(Oxford University Press, 2019) Wany, Aakanksha; Gupta, Alok Kumar; Kumari, Aprajita; Mishra, Sonal; Singh, Namrata; Pandey, Sonika; Vanvari, Rhythm; Igamberdiev, Abir U.; Fernie, Alisdair R.; Gupta, Kapuganti Jagadis
BACKGROUND AND AIMS: Nitrogen (N) levels vary between ecosystems, while the form of available N has a substantial impact on growth, development and perception of stress. Plants have the capacity to assimilate N in the form of either nitrate (NO3-) or ammonium (NH4+). Recent studies revealed that NO3- nutrition increases nitric oxide (NO) levels under hypoxia. When oxygen availability changes, plants need to generate energy to protect themselves against hypoxia-induced damage. As the effects of NO3- or NH4+ nutrition on energy production remain unresolved, this study was conducted to investigate the role of N source on group VII transcription factors, fermentative genes, energy metabolism and respiration under normoxic and hypoxic conditions. METHODS : We used Arabidopsis plants grown on Hoagland medium with either NO3- or NH4+ as a source of N and exposed to 0.8 % oxygen environment. In both roots and seedlings, we investigated the phytoglobin-nitric oxide cycle and the pathways of fermentation and respiration; furthermore, NO levels were tested using a combination of techniques including diaminofluorescein fluorescence, the gas phase Griess reagent assay, respiration by using an oxygen sensor and gene expression analysis by real-time quantitative reverse transcription-PCR methods. KEY RESULTS : Under NO3- nutrition, hypoxic stress leads to increases in nitrate reductase activity, NO production, class 1 phytoglobin transcript abundance and metphytoglobin reductase activity. In contrast, none of these processes responded to hypoxia under NH4+ nutrition. Under NO3- nutrition, a decreased total respiratory rate and increased alternative oxidase capacity and expression were observed during hypoxia. Data correlated with decreased reactive oxygen species and lipid peroxidation levels. Moreover, increased fermentation and NAD+ recycling as well as increased ATP production concomitant with the increased expression of transcription factor genes HRE1, HRE2, RAP2.2 and RAP2.12 were observed during hypoxia under NO3- nutrition. CONCLUSIONS: The results of this study collectively indicate that nitrate nutrition influences multiple factors in order to increase energy efficiency under hypoxia.
Nitrate, NO and ROS signaling in stem cell homeostasis
(Cell Press, 2018) Wany, Aakanksha; Foyer, Christine H.; Gupta, Kapuganti Jagadis
Shoot and root growth is facilitated by stem cells in the shoot and root apical meristems (SAM and RAM). Recent reports have demonstrated a close link between nitrogen nutrition, nitric oxide (NO), and reactive oxygen species (ROS) in the regulation of SAM and RAM functions in response to nitrogen availability.
Nitric oxide is essential for the development of aerenchyma in wheat roots under hypoxic stress
(John Wiley & Sons, 2017) Wany, Aakanksha; Kumari, Aprajita; Gupta, Kapuganti Jagadis
In response to flooding/waterlogging, plants develop various anatomical changes including the formation of lysigenous aerenchyma for the delivery of oxygen to roots. Under hypoxia, plants produce high levels of nitric oxide (NO) but the role of this molecule in plant-adaptive response to hypoxia is not known. Here, we investigated whether ethylene-induced aerenchyma requires hypoxia-induced NO. Under hypoxic conditions, wheat roots produced NO apparently via nitrate reductase and scavenging of NO led to a marked reduction in aerenchyma formation. Interestingly, we found that hypoxically induced NO is important for induction of the ethylene biosynthetic genes encoding ACC synthase and ACC oxidase. Hypoxia-induced NO accelerated production of reactive oxygen species, lipid peroxidation, and protein tyrosine nitration. Other events related to cell death such as increased conductivity, increased cellulase activity, DNA fragmentation, and cytoplasmic streaming occurred under hypoxia, and opposing effects were observed by scavenging NO. The NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt) and ethylene biosynthetic inhibitor CoCl2 both led to reduced induction of genes involved in signal transduction such as phospholipase C, G protein alpha subunit, calcium-dependent protein kinase family genes CDPK, CDPK2, CDPK 4, Ca-CAMK, inositol 1,4,5-trisphosphate 5-phosphatase 1, and protein kinase suggesting that hypoxically induced NO is essential for the development of aerenchyma.
Nitric oxide measurement from purified enzymes and estimation of scavenging activity by gas phase chemiluminescence method
(Springer, 2016) Kumari, Aprajita; Gupta, Alok Kumar; Mishra, Sonal; Wany, Aakanksha; Gupta, Kapuganti Jagadis
In plants, nitrate reductase (NR) is a key enzyme that produces nitric oxide (NO) using nitrite as a substrate. Lower plants such as algae are shown to have nitric oxide synthase enzyme and higher plants contain NOS activity but enzyme responsible for NO production in higher plants is subjected to debate. In plant nitric oxide research, it is very important to measure NO very precisely in order to determine its functional role. A significant amount of NO is being scavenged by various cell components. The net NO production depends in production minus scavenging. Here, we describe methods to measure NO from purified NR and inducible nitric oxide synthase from mouse (iNOS), we also describe a method of measure NO scavenging by tobacco cell suspensions and mitochondria from roots.
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.
Reactive oxygen species, nitric oxide production and antioxidant gene expression during development of aerenchyma formation in wheat
(Taylor & Francis Group, 2018) Wany, Aakanksha; Gupta, Kapuganti Jagadis
In response to hypoxia, plant roots produce very high levels of nitric oxide. Recently, it was demonstrated that NO and ethylene both are essential for development of aerenchyma in wheat roots under hypoxia. Increased NO under hypoxia correlated with induction of NADPH oxidase gene expression, ROS production and lipid peroxidation in cortical cells. Tyrosine nitration was prominent in cells developing aerenchyma suggesting that NO and ROS play a key role in development of aerenchyma. However, the role of antioxidant genes during development of aerenchyma is not known, therefore, we checked gene expression of various antioxidants such as SOD1, AOX1A, APX and MnSOD at different time points after hypoxia treatment and found that expression of these genes elevated in 2 h but downregulated in 24 h where development of aerenchyma is prominent. Further, we found that plants growing under ammonium nutrition displayed delayed aerenchyma development. Taken together, new insights presented in this short communication highlighted additional regulatory role of antioxidants gene expression during aerenchyma development.
The role of nitrite and nitric oxide under low oxygen conditions in plants
(Oxford University Press, 2020) Gupta, Kapuganti Jagadis; Mur, Luis A.J.; Wany, Aakanksha; Kumari, Aprajita; Fernie, Alisdair R.; Ratcliffe, R. George
Plant tissues, particularly roots, can be subjected to periods of hypoxia due to environmental circumstances. Plants have developed various adaptations in response to hypoxic stress and these have been extensively described. Less well-appreciated is the body of evidence demonstrating that scavenging of nitric oxide (NO) and the reduction of nitrate/nitrite regulate important mechanisms that contribute to tolerance to hypoxia. Whilst ethylene controls hyponasty and aerenchyma formation, NO production apparently regulates hypoxic ethylene biosynthesis. In the hypoxic mitochondrion, cytochrome c oxidase, which is a major source of NO, is also inhibited by NO, thereby reducing the respiratory rate and enhancing local oxygen concentrations. Nitrite can maintain ATP generation under hypoxia by coupling its reduction to the translocation of protons from the inner side of mitochondria and generating an electrochemical gradient. This reaction can be further coupled to a reaction whereby non-symbiotic haemoglobin oxidizes NO to nitrate. In addition to these functions, nitrite has been reported to influence mitochondrial structure and supercomplex formation, as well as playing a role in oxygen sensing via the N-end rule pathway. These studies establish that nitrite and NO perform multiple functions during plant hypoxia and suggest that further research into the underlying mechanisms is warranted.