Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves

Abstract

Studies were conducted to quantitate the evolution of nitrogen oxides (NO(.)) from soybean GlGycine max (L.) Merr.j leaves during in vivo nitrate reductase (NR) assays with aerobic and anaerobic gas purging. Anaerobic gas purging (N2 and argon) consistently resulted in greater NO(.) evolution than did aerobic gas purging (air and 02). The evolution of NO(.) was dependent on gas flow rate and on N02-formation in the assay medium; although a threshold level of N02-appeared to exist beyond which the rate of NO(.) evolution did not increase further. (L.) Merr.] the in vivo NR assay has been optimized for leaves (10,13,17) and roots (7), and the assay has been used to compare estimated reduced N input with actual (Kjeldahl) reduced N accumulation (7, 9, 11). One notable difference between optimized in vivo NR assays of soybean roots and leaves was that roots required N2 purging during assay (7), while N2 purging of leaves during assay resulted in inconsistent results, ranging from slight stimulation to marked inhibition of nitrite accumulation (unpublished data).It has been shown that air purging of herbicide-treated soybean leaves results in evolution of NO and NO2 (collectively NO(.)) (16). The reaction mechanism(s) leading to NO(.) evolution are unknown, although Klepper (16) has proposed that accumulated N02 reacts with plant metabolite(s) to form NO and NO2.Nitrogen oxide (NO) seems to be the primary gas form evolved (16), since NO2 is known to be readily soluble in aqueous solution, forming N02-and NO3&; the equilibrium of the latter two ions being dependent on solution conditions (1).The above observations suggested that similar evolution of NO(x) gasses may be occurring during gas purging of the in vivo l Abbreviations: NR, nitrate reductase; NO(X), refers collectively to nitric oxide (NO) and nitrogen dioxide (NO2); DAP, days after planting. NR assay medium. In addition, it was expected that N2, air, and 02 purging of the in vivo NR assay medium would result in differential accumulation of N02-, due to a more complete blockage of further reduction of N02-to NH4+ under more anaerobic conditions. The objectives of this study were to (a) establish why N2 purging of the in vivo NR assay medium resulted in inconsistent N02 accumulation relative to no gas purging, and (b) determine if evolution of NO(.) gasses under anaerobic and aerobic gas purging of the in vivo NR assay medium could account for the decrease in nitrite accumulation during NR assays of physiologically young soybean leaves. Instruments Co., Lincoln, NE) for a 14-h light period at 27 to 30°C; nighttime temperatures ranged from 17 to 21°C. Plants were sampled at intervals from 11 to 25 DAP for analysis of leaf NR activity. Twenty seedlings were harvested at each sampling time and leaflets from respective nodes were composited and subsampled, 20 leaf discs (1 cm diameter) for each sample (=0.2 g tissue). MATERIALS AND METHODS Plant Growth and SamplingGrowth

Nitrite–dependent nitric oxide production pathway: implications for involvement of active nitrogen species in photoinhibitionin vivo

“…The ¢rst clue was provided by Harper in 1981 who demonstrated the e¡ects of gas purging on the in vivo NR assay (Harper 1981). This assay is a technique to determine the nitrate-reducing activity of tissues (Nicholas et al 1976).…”

“…A strong correlation between NOx evolution and NO À 2 accumulation was observed. Since no NOx evolution occurred in boiled (heat-denatured) leaf disks, Harper presumed an enzymatic NOx evolution pathway from NO À 2 in leaves (Harper 1981). Using a chlorate screening procedure, Harper and others isolated soybean mutants that lacked the constitutive NR activity in leaves but retained inducible NR activity in response to NO 3 Ryan et al 1983).…”

“…Nitric oxide (NO) and nitric dioxide (NO 2 ) have been reported to be produced in leaves of higher plants (Dean & Harper 1986;Harper 1981;Klepper 1990). However, the mechanism of the active nitrogen production and its e¡ects on photosynthesis are largely unknown.…”

Nitrate reductase activity in leaves and stems of tanner grass (Brachiaria radicans Napper)

“…The in vitro assay is accomplished by extracting the enzymatic fraction from the tissue (Lillo, 1983;Viégas et al, 1999), and by measuring the reaction in an appropriate buffered medium (Aslam et al, 1984;Lillo, 1983). On the other hand, the in vivo assay can be carried out easily, without enzyme extraction, by incubating tissue samples in a buffered medium containing NO 3 -, solvent and/or detergent, and by measuring NO 2 -produced during an adequate period of time, in the dark (Jaworski, 1971;Jones & Sheard, 1977;Harper, 1981). Nitrite production in the in vivo assay depends on the diffusion of NO 3 -into the tissue, to reach the cytoplasm of cells where the enzyme is located.…”

The Role of Nitric Oxide in Plants

“…In addition to the putative NOS pathway, nitrate reductase (NR) may produce NO in plants. NR, a key enzyme for nitrate assimilation in higher plants that catalyzes the conversion of nitrate to nitrite, was shown to produce NO from nitrite by employing NAD(P)H at an optimum pH of 6.75 both under in vitro and in vivo conditions (15,30,63). Table 1 summarizes the physiological and biochemical processes that have been shown to be affected by NO in plants (7).…”