Browsing by Author "Gupta, Alok Kumar"

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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.
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.
Moving nitrogen to the centre of plant defence against pathogens
(Oxford University Press, 2017) Mur, Luis A.J.; Simpson, Catherine; Kumari, Aprajita; Gupta, Alok Kumar; Gupta, Kapuganti Jagadis
Background Plants require nitrogen (N) for growth, development and defence against abiotic and biotic stresses. The extensive use of artificial N fertilizers has played an important role in the Green Revolution. N assimilation can involve a reductase series (NO–3→ NO–2 → NH+4) followed by transamination to form amino acids. Given its widespread use, the agricultural impact of N nutrition on disease development has been extensively examined. Scope: When a pathogen first comes into contact with a host, it is usually nutrient starved such that rapid assimilation of host nutrients is essential for successful pathogenesis. Equally, the host may reallocate its nutrients to defence responses or away from the site of attempted infection. Exogenous application of N fertilizer can, therefore, shift the balance in favour of the host or pathogen. In line with this, increasing N has been reported either to increase or to decrease plant resistance to pathogens, which reflects differences in the infection strategies of discrete pathogens. Beyond considering only N content, the use of NO-3 or NH+4 fertilizers affects the outcome of plant–pathogen interactions. NO-3 feeding augments hypersensitive response- (HR) mediated resistance, while ammonium nutrition can compromise defence. Metabolically, NO–3 enhances production of polyamines such as spermine and spermidine, which are established defence signals, with NH+4 nutrition leading to increased γ-aminobutyric acid (GABA) levels which may be a nutrient source for the pathogen. Within the defensive N economy, the roles of nitric oxide must also be considered. This is mostly generated from NO–2 by nitrate reductase and is elicited by both pathogen-associated microbial patterns and gene-for-gene-mediated defences. Nitric oxide (NO) production and associated defences are therefore NO-3 dependent and are compromised by NH+4. Conclusion:This review demonstrates how N content and form plays an essential role in defensive primary and secondary metabolism and NO-mediated events.
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.
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.