Recommendations of using at least two different methods for measuring NO

Nitric oxide (NO) is widely recognized as a signal molecule in plants. Various sources of NO were identified in plants (Moreau et al., 2010; Gupta et al., 2011; Mur et al., 2013) located in different compartments activated under various conditions. Briefly these are the mitochondrial nitrite: NO reductase reaction, the cytosolic nitrate reductase (NR), the plasma membrane nitrite: NO reductase (PM-NiNOR), xanthine oxidoreductase, NO synthase-like enzyme (putative), polyamine (PA)- and hydroxylamine (HA)-mediated pathways. NO acts as an intracellular messenger due to its diffusible capacity through various cellular compartments. After pioneering discovery of NO production in plants, scientists started digging deeply to the key function of NO and this research led to understanding of various roles of NO that include regulation of stomatal movement, root development, floral transition, response to biotic and abiotic stresses, symbiotic interactions. Despite of extensive research on NO roles in metabolism and signal transduction, its measurement remains challenging (Vandelle and Delledonne, 2008; Mur et al., 2011). We discuss below the problems associated with NO measurement and suggest some important solutions to tackle these problems. The half-life of NO depends on its concentration and usually falls is in the range of ten(s) seconds. For instance, at 10 μM concentration NO has a half-life of about 80 s whereas at 100 μM concentration NO has a half-life of about 8 s (Wink and Mitchell, 1998). This means that at low concentration NO can easily diffuse from its origin to the site of action. At higher concentrations most of the NO rapidly undergoes autoxidation. Moreover NO half-life also depends on the presence of scavengers such as non-symbiotic hemoglobin (class 1) and other major NO scavenging targets such as lipids and metal-containing proteins. Therefore, it is often very important to measure NO concentration which is crucial for understanding its function. But NO concentrations measured in same biological material by different methods often give different values (Mur et al., 2006; Planchet and Kaiser, 2006). In some cases NO production is localized to specific cells such as guard cells; therefore fluorescent probes are required to visualize NO producing sites. Here we describe why we need to use at least two different methods for measuring generation of NO in plants and suggest the best combination of methods for specific studies.