Browsing by Author "Bulle, Mallesham"

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Alternative oxidase is an important player in the regulation of nitric oxide levels under normoxic and hypoxic conditions in plants
(Oxford University Press, 2019) Kumari, Aprajita; Pathak, Pradeep Kumar; Bulle, Mallesham; Igamberdiev, Abir U; Gupta, Kapuganti Jagadis
Plant mitochondria possess two different pathways for electron transport from ubiquinol: the cytochrome pathway and the alternative oxidase (AOX) pathway. The AOX pathway plays an important role in stress tolerance and is induced by various metabolites and signals. Previously, several lines of evidence suggested that the AOX pathway prevents overproduction of superoxide and other reactive oxygen species. More recent evidence suggests that AOX also plays a role in regulation of nitric oxide (NO) production and signalling. The AOX pathway is induced under low phosphate, hypoxia, pathogen infections and elicitor treatments. The induction of AOX under aerobic conditions in response to various stresses can reduce electron transfer through complexes III and IV and thus prevents the leakage of electrons to nitrite and the subsequent accumulation of NO. Excess of NO under various stresses can inhibit complex IV, thus the AOX pathway minimizes nitrite-dependent NO synthesis that would arise from enhanced electron leakage in the cytochrome pathway. By preventing NO generation, AOX can reduce peroxynitrite formation and tyrosine nitration. In contrast to its function under normoxia, AOX has a specific role under hypoxia, where AOX can facilitate nitrite-dependent NO production. This reaction drives the phytoglobin-NO cycle to increase energy efficiency under hypoxia.
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.
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 Jagadis
Isolation 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.
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.
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.
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.
Polyamine induction in postharvest banana fruits in response to NO donor SNP occurs via L-arginine mediated pathway and not via competitive diversion of S-adenosyl-L-methionine
(MDPI AG, 2019) Lokesh, Veeresh; Manjunatha, Girigowda; Hegde, Namratha S.; Bulle, Mallesham; Puthusseri, Bijesh; Gupta, Kapuganti Jagadis; Neelwarne, Bhagyalakshmi
Nitric oxide (NO) is known to antagonize ethylene by various mechanisms; one of such mechanisms is reducing ethylene levels by competitive action on S-adenosyl-L-methionine (SAM)—a common precursor for both ethylene and polyamines (PAs) biosynthesis. In order to investigate whether this mechanism of SAM pool diversion by NO occur towards PAs biosynthesis in banana, we studied the effect of NO on alterations in the levels of PAs, which in turn modulate ethylene levels during ripening. In response to NO donor sodium nitroprusside (SNP) treatment, all three major PAs viz. putrescine, spermidine and spermine were induced in control as well as ethylene pre-treated banana fruits. However, the gene expression studies in two popular banana varieties of diverse genomes, Nanjanagudu rasabale (NR; AAB genome) and Cavendish (CAV; AAA genome) revealed the downregulation of SAM decarboxylase, an intermediate gene involved in ethylene and PA pathway after the fifth day of NO donor SNP treatment, suggesting that ethylene and PA pathways do not compete for SAM. Interestingly, arginine decarboxylase belonging to arginine-mediated route of PA biosynthesis was upregulated several folds in response to the SNP treatment. These observations revealed that NO induces PAs via l-arginine-mediated route and not via diversion of SAM pool.