Initiation of nitric oxide (NO) synthesis in roots of etiolated seedlings of pea (Pisum sativum L.) under the influence of nitrogen-containing compounds

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.