Limits...
Limits in the use of cPTIO as nitric oxide scavenger and EPR probe in plant cells and seedlings.

D'Alessandro S, Posocco B, Costa A, Zahariou G, Schiavo FL, Carbonera D, Zottini M - Front Plant Sci (2013)

Bottom Line: Despite its recognized biological role, the sensitivity and effectiveness of the methods used for measuring NO concentration in plants are still under discussion.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padova Padova, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

Electron paramagnetic resonance signal of cPTIO incubated in water, PBS, exhausted medium or with dead cells. cPTIO(NNO) was incubated in water, PBS, exhausted medium (EM) or with boiled dead cells (DC). EPR spectra of the samples were detected after 180 min of incubation. Intensities of EPR signals are given as percentage of the total signal at t0.
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Figure 3: Electron paramagnetic resonance signal of cPTIO incubated in water, PBS, exhausted medium or with dead cells. cPTIO(NNO) was incubated in water, PBS, exhausted medium (EM) or with boiled dead cells (DC). EPR spectra of the samples were detected after 180 min of incubation. Intensities of EPR signals are given as percentage of the total signal at t0.

Mentions: In order to verify whether the reduction of cPTIO EPR signal was associated with the presence of a cell-linked activity, EPR measurements were performed incubating cPTIO(NNO) either in exhausted culture medium, withdrawn from 5-day-old cell cultures, or in the presence of boiled 5-day-old cell cultures. In both cases, the intensity of EPR signals was maintained for longer time compared with the previous experiments, with a signal decrease of less than 10% after 180 min (Figure 3).


Limits in the use of cPTIO as nitric oxide scavenger and EPR probe in plant cells and seedlings.

D'Alessandro S, Posocco B, Costa A, Zahariou G, Schiavo FL, Carbonera D, Zottini M - Front Plant Sci (2013)

Electron paramagnetic resonance signal of cPTIO incubated in water, PBS, exhausted medium or with dead cells. cPTIO(NNO) was incubated in water, PBS, exhausted medium (EM) or with boiled dead cells (DC). EPR spectra of the samples were detected after 180 min of incubation. Intensities of EPR signals are given as percentage of the total signal at t0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756283&req=5

Figure 3: Electron paramagnetic resonance signal of cPTIO incubated in water, PBS, exhausted medium or with dead cells. cPTIO(NNO) was incubated in water, PBS, exhausted medium (EM) or with boiled dead cells (DC). EPR spectra of the samples were detected after 180 min of incubation. Intensities of EPR signals are given as percentage of the total signal at t0.
Mentions: In order to verify whether the reduction of cPTIO EPR signal was associated with the presence of a cell-linked activity, EPR measurements were performed incubating cPTIO(NNO) either in exhausted culture medium, withdrawn from 5-day-old cell cultures, or in the presence of boiled 5-day-old cell cultures. In both cases, the intensity of EPR signals was maintained for longer time compared with the previous experiments, with a signal decrease of less than 10% after 180 min (Figure 3).

Bottom Line: Despite its recognized biological role, the sensitivity and effectiveness of the methods used for measuring NO concentration in plants are still under discussion.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Padova Padova, Italy.

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.

No MeSH data available.


Related in: MedlinePlus