Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay

Abstract

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N20) were identified by GC and GC/MS as the main N products formed. NO and N20 produced from soybean leaflets were both labeled with 5N when "5N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N20 were identified as air (N2, 02), C02, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N20 (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N20 were not evolved from leaves of the nrl soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO(x) (NO and NO2). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Meff., and Pueraria thunbergiana Benth. evolved lower levels of NO(,).Klepper (5) demonstrated that herbicide treated soybean leaves form and release NO(X3 (thought to be predominately NO). He suggested that NO and NO2 were formed by a chemical reaction of NO2-(accumulated due to herbicide treatments) with plant metabolites, forming NO at low NO2-levels and NO and NO2 at higher NO2 levels. It was later shown (4) that N2 gas purging during the in vivo NR assay of soybean leaflets also resulted in the formation of NO(X) derived from NO2-accumulated during the assay. Harper's work (4) also indicated that the predominate ' Supported by an American Soybean Association research grant, project number 84953.2 Grateful recipient of a Wright Fellowship from the College of Agriculture, University of Illinois.3 Abbreviations: NO(X), refers collectively to nitric oxide (NO) and nitrogen dioxide (NO2); NR, nitrate reductase; DAP, days after planting; N20, nitrous oxide; m/z, mass to charge ratio; bp, boiling point. compound evolved was NO, but based on results with boiled leaf discs he implied that an enzymic reaction was responsible for the NO(x) evolution. Additional evidence for an enzymic reaction was provided by the isolation of a mutant soybean line (nr...

Emission of nitrous oxide (N2O) from transgenic tobacco expressing antisense NiR mRNA

“…We investigated the emission of N 2 or N 2 O from intact tobacco clone 271, which expresses antisense NiR mRNA (Vaucheret et al, 1992) Dean and Harper (1986) reported that 90 nmole N 2 O g ±1 FW h ±1 of N 2 O was emitted from soybean. The difference in the N 2 O emission rates of soybean and clone 271 tobacco may be due to differences in the experimental systems used or differences in the source of N 2 O in the plants.…”

“…The possibility that micro-organisms participated in the denitri®cation could not therefore be excluded. Soybean and winged bean have been reported to emit NOx (NO and NO 2 ) and N 2 O during helium-purged in vivo nitrate reductase (NR) assay of leaf discs (Dean and Harper, 1986). It is not clear whether intact plants can denitrify under normal growth conditions.…”

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

“…It was later determined that there were two forms of constitutive NR in wild-type soybean leaves, which were designated c 1 NR and c 2 NR (Streit et al 1985). Although there was controversy over the NOx evolution pathway from NO 3 via NO (Mulvaney & Hageman 1984), evidence that NO and N 2 O were produced from NO 3 during nitrate reduction was provided by gas chromatography, mass spectrometry and 15 N tracer techniques (Dean & Harper 1986). Because the c 1 NAD(P)H:NR (EC 1.6.6.2) is unique to the Phaseoleae tribe of the family Leguminosae (Dean & Harper 1986), the evolution of NOx through the NR-dependent pathway was thought to be limited to legume species among higher plants (Dean & Harper 1988).…”

“…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.…”

Wheat leaves emit nitrous oxide during nitrate assimilation

“…Leaf N2O emissions were correlated with leaf nitrate assimilation activity, as measured by using the assimilation quotient, the ratio of CO2 assimilated to O2 evolved. 15 P lants play a critical role in regulating the chemical and physical state of the atmosphere through the exchange of biogenic greenhouse gases. Most notable are plant-atmosphere exchanges of CO 2 , O 2 , and H 2 O, but leaves also emit a variety of carbon-and nitrogen-based trace gases involved in climate alteration processes (1).…”