Abstract
Over the last decade the importance of nitric oxide (NO) in plant signaling has emerged. Despite its recognized biological role, the sensitivity and effectiveness of the methods used for measuring NO concentration in plants are still under discussion. Among these, electron paramagnetic resonance (EPR) is a well-accepted technique to detect NO. In the present work we report the constraints of using 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in biological samples as spin trap for quantitative measurement of NO. EPR analyses on Arabidopsis cell cultures and seedlings show that cPTIO(NNO) is degraded in a matter of few minutes while the (INO) compound, produced by cPTIO and NO reaction, has not been detected. Limitations of using this spin trap in plant systems for quantitative measurements of NO are discussed. As NO scavenger, cPTIO is widely used in combination with 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescent dye in plant research. However, the dependence of DAF-FM fluorescence on cPTIO and NO concentrations is not clearly defined so that the range of concentrations should be tightly selected. In this context, a systematic study on cPTIO NO scavenging properties has been performed, as it was still lacking for plant system applications. The results of this systematic analysis are discussed in terms of reliability of the use of cPTIO in the quantitative determination and scavenging of NO in plants and plant cultured cells.
Highlights
Nitric oxide (NO) is a signal molecule involved in controlling both physiological processes and stress responses (Mur et al, 2013)
To assess the spin trap stability in the presence of biological samples, a series of experiments were performed in vivo on Arabidopsis cultured cells, by incubating 5-day-old cell cultures with 100 μM cPTIO(NNO) or cPTIO(INO)
It was observed that the intensity of electron paramagnetic resonance (EPR) signals of both cPTIO(NNO) and cPTIO(INO) rapidly decreased in the first minutes of incubation, reaching nearly zero after 130 min (Figure 2)
Summary
Nitric oxide (NO) is a signal molecule involved in controlling both physiological processes and stress responses (Mur et al, 2013) It plays an important role in root organogenesis and development (Correa-Aragunde et al, 2004) and in auxin signaling (Kramer and Bennett, 2006) and perception (Terrile et al, 2012). The controversial existence of NO synthaselike enzymes makes it difficult to define the specific NO source engaged in a specific physiological process and to understand how it is involved in it. For this reason, in order to establish whether and where NO is produced by specific cells and tissues, plant researchers rely on several indirect methods of analysis. In biological systems, the use of these methods is limited by the short half-life of the molecule (Woldman et al, 1994; Gupta and Igamberdiev, 2013)
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