Publications of NIPGR Scientists
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Item The emerging roles of nitric oxide and its associated scavengers-phytoglobins-in plant symbiotic interactions(Oxford University Press, 2024) Pathak, Pradeep Kumar; Yadav, Nidhi; Kaladhar, Vemula Chandra; Jaiswal, Rekha; Kumari, Aprajita; Igamberdiev, Abir U.; Loake, Gary J.; Gupta, Kapuganti JagadisA key feature in the establishment of symbiosis between plants and microbes is the maintenance of the balance between the production of the small redox-related molecule, nitric oxide (NO), and its cognate scavenging pathways. During the establishment of symbiosis, a transition from a normoxic to a microoxic environment often takes place, triggering the production of NO from nitrite via a reductive production pathway. Plant hemoglobins [phytoglobins (Phytogbs)] are a central tenant of NO scavenging, with NO homeostasis maintained via the Phytogb–NO cycle. While the first plant hemoglobin (leghemoglobin), associated with the symbiotic relationship between leguminous plants and bacterial Rhizobium species, was discovered in 1939, most other plant hemoglobins, identified only in the 1990s, were considered as non-symbiotic. From recent studies, it is becoming evident that the role of Phytogbs1 in the establishment and maintenance of plant–bacterial and plant–fungal symbiosis is also essential in roots. Consequently, the division of plant hemoglobins into symbiotic and non-symbiotic groups becomes less justified. While the main function of Phytogbs1 is related to the regulation of NO levels, participation of these proteins in the establishment of symbiotic relationships between plants and microorganisms represents another important dimension among the other processes in which these key redox-regulatory proteins play a central role.Item An efficient method of mitochondrial DNA isolation from Vigna radiata for genomic studies(Springer Nature Publishing AG, 2020) Singh, Pooja; Sahoo, Ranjan Kumar; Bulle, Mallesham; Gupta, Kapuganti JagadisIsolation of mitochondrial DNA from root tissues of mung bean (Vigna radiata) is quite tedious, complex, and often results in low yield. Hence here we show a simple, rapid, and improved protocol for isolation of mitochondrial DNA from root tissues of hydroponically grown mung bean plants. This method involves purification of mitochondria and subsequent isolation of DNA from obtained purified mitochondria. For this purpose, mitochondria were isolated using a discontinuous Percoll gradient centrifugation followed by RNase I treatment to the isolated DNA to remove any traces of RNA contamination. The mitochondrial DNA was isolated from mitochondrial samples by commonly used CTAB method. The specificity of isolated mitochondrial DNA was confirmed using mtDNA-specific genes (NAD1 and COX3). β-Actin primer was used to check the nuclear DNA contamination. PCR amplification was observed in mtDNA specific genes NAD1 and COX3 except nuclear encoded β-actin gene suggesting that mitochondrial DNA was not contaminated by nuclear DNA.Item Isolation of physiologically active and intact mitochondria from chickpea(Springer, 2017) Pandey, Sonika; Kumari, Aprajita; Gupta, Kapuganti JagadisChickpea is an important leguminous crop that belongs to Fabaceae family, highly valued for its nutritious seeds. Seeds contain reserve food for the developing embryos. Mitochondria are crucial organelle for generation of chemical energy in the form of ATP which is required for achieving metabolically active state; therefore, investigating mitochondrial function and respiration rate is crucial for exploring various metabolic and physio-biochemical changes that occur during seed germination. Here we describe a method for isolation of mitochondria from germinating seeds of two chickpea varieties, i.e., Desi and Kabuli. Structure of Mitotracker-stained isolated mitochondria was observed by confocal microscopy and respiration rate was measured using an oxygen microsensor.Item Isolation and structural studies of mitochondria from pea roots(Springer, 2017) Vishwakarma, Abhaypratap; Gupta, Kapuganti JagadisFor structural and respiratory studies, isolation of intact and active mitochondria is essential. Here, we describe an isolation method which gave good yield and intact mitochondria from 2-week-old pea (Pisum sativum) roots grown hydroponically under standard growth conditions. We used Percoll gradient centrifugation for this isolation procedure. The yield of purified mitochondria was 50 μg/g FW. Isolated mitochondria maintained their structure which was observed by using MitoTracker green in confocal microscope and scanning electron microscopy (SEM). Intact mitochondria are clearly visible in SCM images. Taken together this isolation method can be used for physiological and microscopic studies on mitochondria.Item 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 JagadisNO 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.Item 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 JagadisIn 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.Item 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 JagadisIn 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.