Hemoglobin as a probe for estimation of nitric oxide emission from plant tissues
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METHODOLOGY
Hemoglobin as a probe for estimation of nitric oxide emission from plant tissues Neha Singh and Satish C. Bhatla*
Abstract Background: Plant roots contribute significant amount of nitric oxide (NO) in the rhizosphere as a component of NO in the ecosystem. Various pharmacological investigations on NO research in plants seek to quench endogenous NO by using externally applied NO quenchers, mainly 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) and its more soluble form-carboxy-PTIO (cPTIO). Owing to serious limitations in its application cPTIO is no more a desired compound for such applications. Result: Present work highlights the significance of using hemoglobin in the bathing solution to not only release endogenous NO from plant tissue but also to quench it in a concentration-dependent manner. Conclusion: The protocol further demonstrates the diffusibility of NO from intracellular locations in presence of externally provided hemoglobin. The proposed method can have widespread applications as a substitute to debatable and currently used cPTIO as a NO scavenger. Keywords: Hemoglobin, Nitric oxide, NO scavenger, Methemoglobin, MnIP-Cu Background Plants constitute an important source of biological NO emission in the terrestrial ecosystem [1, 2]. Following the first observation of NO emission from herbicide-treated soybean leaves into the atmosphere [3], several studies have focused on NO emissions from detached plant tissues, cell suspensions and mitochondria [2, 4–7]. NO is a gaseous, lipophilic biomolecule which acts as a free radical with ability to diffuse across cell membranes, through the cytoplasm and migrate intracellularly as well as from cell to cell across the apoplast. It diffuses at a rate of 50 μm s−1. Its solubility is 1.9 mM in aqueous solutions at 1 atm pressure. Half-life of NO in biological systems is reasonably short, less that 10 s. The rapid movement and removal of cellular NO, makes it an ideal signaling molecule for cell to cell communication in plant tissues both in normal growth conditions and under stress [3, 8–12]. It is a versatile molecule that can migrate and act concurrently in different cellular compartments and in *Correspondence: [email protected] Laboratory of Plant Physiology and Biochemistry, Department of Botany, University of Delhi, Delhi 110007, India
opposite directions. NO is biosynthesized in plants via multiple routes which are broadly classified as reductive and oxidative pathways. It is produced through both enzymatically in plastids, mitochondria, chloroplasts, and non-enzymatically in the apoplast [6, 13–19]. Pharmacological investigations on the modulation of plant growth and development by NO routinely employ PTIO and its more soluble form-cPTIO as a means to quench tissue NO. Of late, it has been reported that cPTIO usage as a NO scavenger exhibits duality in its action [20]. Depending on concentration, cPTIO can, at times, even contribute to further NO production, rather than serving as a NO quencher. cPT
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