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Detection of Nitric Oxide in Plants by Electron Spin Resonance

Three methods to detect nitric oxide (NO^•) are reported here. The first method was determining NO^• in extracted plant tissue. NO^• was trapped by spin trapping reagent containing diethyldithiocarbamate (DETC) and FeSO₄, extracted by ethyl acetate, and determined with an electron spin resonance (ESR) spectrometer. The second method was indirectly determining NO^• in live wheat leaves. Seedlings were cultured in a medium containing FeSO₄, and the leaves were brushed by DETC. Then, the leaves were ground and the complex of (DETC)₂-Fe²⁺-NO was extracted and determined with an ESR spectrometer. The third method was directly determining NO• in live wheat leaves. After treating plant materials as in the second method, part of the water in leaves was transpired, and the leaf disks were inserted directly into quartz tubes to determine NO^• with an ESR spectrometer. The NO_• scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline- 1-oxyl 3-oxide (PTIO) decreased NO^• signal detected either by an indirect or a direct method. This result indicates that both methods could detect NO^• in the live plant. Using the first methods, we detected NO^• change in wheat infected by Puccinia striiformis race CY22-2 pathogen (incompatible interaction) at different inoculation times, and it was found that the NO^• content dramatically increased at 24 h postinoculation, quickly decreased at 48 h, and increased again at 96 h.