Received December 26, 2012; Revision received January 24, 2013
The level of nitric oxide (NO) in roots of 2-day-old etiolated pea (Pisum sativum L.) seedlings was investigated by fluorescence microscopy using the fluorescent probe 4,5-diaminofluorescein diacetate. Segments representing transversal (cross) cuts of the roots having thickness of 100 to 150 µm (a segment of the root located 10 to 15 mm from the apex) were analyzed. A substantial concentration of NO in the roots was registered when the seedlings were grown in water (control). Addition of 4 mM sodium nitroprusside, 20 mM KNO3, 2 mM NaNO2, 2 mM L-arginine into the growth medium increased NO concentration with respect to the control by 1.7- to 2.3-fold. Inhibitors of animal NO-synthase – 1 mM Nω-nitro-L-arginine methyl ester hydrochloride and 1 mM aminoguanidine hydrochloride – reduced the intensity of fluorescence in the root segments in the presence of all the studied compounds. In medium with KNO3, the inhibitor of nitrate reductase –150 µM sodium tungstate – lowered the fluorescence intensity by 60%. Scavengers of nitric oxide – 100 µM 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and 4 µM hemoglobin – lowered NO concentration in all the studied variants. Potassium ferrocyanide (4 mM) as the inactive analog of sodium nitroprusside inhibited generation of NO. These results are discussed regarding possible pathways of NO synthesis in plants.
KEY WORDS: Pisum sativum L., etiolated seedlings of pea, fluorescence probe, nitric oxide (NO), inhibitors of animal NO-synthase (NOS) and plant nitrate reductase, NO scavengers