Nitric Oxide and Nitrous Oxide Production by Soybean andWinged Bean during the in Vivo Nitrate Reductase Assay'
Received for publication April 1, 1986 and in revised form July 15, 1986
JOHN V. DEAN2 AND JAMES E. HARPER*Department ofAgronomy (J.V.D.) and United States Department ofAgriculture, Agricultural ResearchService (J.E.H.), University of Illinois, Urbana, Illinois 61801
ABSTRACT
This study was conducted to determine by gas chromatography (GC)and mass spectrometry (MS) the identity and the quantity of volatile Nproducts produced during the helium-purged in vivo NR assay of soybean(Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpustetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identi-fication and quantitation was collected by cryogenic trapping of volatilecompounds carried in the He-purge gas stream. As opposed to an earlierreport, acetaldehyde oxime production was not detected by our GCmethod, and acetaldehyde oxime was shown to be much more soluble inwater than the compound(s) evolved from soybean leaflets. Nitric oxide(NO) and nitrous oxide (N20) were identified by GC and GC/MS as themain N products formed. NO and N20 produced from soybean leafletswere both labeled with 5N when "5N-nitrate was used in the assaymedium, demonstrating that both were produced from nitrate duringnitrate reduction. Other compounds co-trapped with NO and N20 wereidentified as air (N2, 02), C02, methanol, acetaldehyde, and ethanol.Leaves of winged bean, subjected to the purged in vivo NR assay, evolvedgreater quantities of NO and N20 (13.9 and 0.37 micromole per gramfresh weight per 30 minutes, respectively) than did the soybean cvWilliams (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 thenrl soybean mutant which lacks the constitutive NR enzymes. In additionto soybean cv Williams, six other Glycine sp. examined evolved signifi-cant quantities of NO(x) (NO and NO2). Other species including Neono-tonia 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 leavesform and release NO(X3 (thought to be predominately NO). Hesuggested that NO and NO2 were formed by a chemical reactionof NO2- (accumulated due to herbicide treatments) with plantmetabolites, forming NO at low NO2- levels and NO and NO2at higher NO2 levels. It was later shown (4) that N2 gas purgingduring the in vivo NR assay of soybean leaflets also resulted inthe formation of NO(X) derived from NO2- accumulated duringthe 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 Agri-culture, University of Illinois.
3 Abbreviations: NO(X), refers collectively to nitric oxide (NO) andnitrogen 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 leafdiscs he implied that an enzymic reaction was responsible forthe NO(x) evolution. Additional evidence for an enzymic reactionwas provided by the isolation of a mutant soybean line (nr1) thatlacked both NO(x) evolution and the constitutive NR enzymes(11, 14).
Subsequently, other workers (10) confirmed that significantquantities of a N compound were evolved during the purged invivo NR assay of soybean leaflets. However, using MS, UVspectroscopy, and '5N-labeled nitrate, NO was not detected. They(10) concluded that acetaldehyde oxime was the majorN productevolved and that this compound was responsible for the previousresults (4) which were obtained with methods that were notspecific for the detection of NO. Preliminary work based on thedifferential water solubilities of acetaldehyde oxime and com-pound(s) evolved from soybean leaflets, and our inability todetect acetaldehyde oxime by GC methods as a compoundevolving from soybean leaflets, led us to reevaluate the com-pound(s) evolved. The purposes of this study were, therefore, to:(a) reevaluate, by GC, GC/MS, and '5N techniques, the identityof the major compound evolved during the in vivo NR assay ofsoybean leaflets; (b) quantitate the amount of the N compoundevolved; and (c) screen other species for the ability to evolve thisN gas.
MATERIALS AND METHODS
Plant Material. Wild type (cv Williams) and nr1 mutant soy-bean seeds (Glycine max [L.] Merr.) were germinated and grownas previously described by Harper (4) for growth chamber studies,except that the quantum flux supplied by incandescent andfluorescent lamps was 350 ,E m-2 s-' (measured with an LI-185A quantum sensor,4 Lambda Instruments Co., Lincoln, NE).Seeds were germinated in sand trays and watered daily withdeionized H20 until 7 DAP, followed by daily watering with acomplete Hoagland nutrient solution, containing 15 mm NO3.At 10 DAP, the unifoliolate leaves were harvested 4 h after theonset of illumination and immediately prepared for the assays.Plants used for '5N studies were germinated and grown as aboveexcept that the nutrient solution did not contain NO3-. This wasdone to eliminate interference by 14N-NO3- in the tissue withthe '5N-NO3- supplied during the assay.
4Mention of a trademark, vendor, or proprietary product does notconstitute a guarantee or warranty of the vendor or product by theUnited States Department ofAgriculture, and does not imply its approvalto the exclusion of other vendors or products that may also be suitable.
718 www.plantphysiol.orgon April 3, 2019 - Published by Downloaded from
Hymowitz, Glycine canescens F. J. Herm, Glycine tabacina(Labill.) Benth., Glycine soja Sieb. and Zucc., and Glycine clan-destina Wendl., were germinated and grown as described aboveexcept that the sand trays were watered with the completenutrient solution (15 mM NO3-) beginning at 9 DAP and theplants were harvested at 13 DAP.
Solubility of N Compound(s) Evolved from Soybean Leaflets,Acetaldehyde Oxime, and NO. To test the solubility of the Ncompound(s) evolved from soybean leaflets, the NO(x) assaysystem described by Harper (4) was modified in the followingmanner: after the N2 purge gas stream was bubbled (150 mlmin-') through 10 ml of buffer solution containing leaf discs, itwas bubbled through 20 ml of water and then through theoxidizer column and trapping solution. The solubility ofauthen-tic acetaldehyde oxime was tested in the same manner exceptthat 10 mM acetaldehyde oxime replaced the nitrate and leafdiscs in the buffer solution. The solubility of authentic NO wastested by injecting an aliquot of NO directly into the purge gasstream before the water trap.GC Analysis of NO, N20, C02, and Air. Unifoliolate leaves
(15 g) of soybean, the nr, mutant, and winged bean were slicedperpendicular to the midrib into about 1 cm strips and placedinto a 1 L foil-covered flask containing 500 ml of buffer (0.10 MK2HPO4-KH2PO4 [pH 7.5]; 0.05 M KNO3). The leaf strips werevacuum infiltrated twice with buffer (2 min each time). The flaskand contents were attached to the all-glass system (except for thepressure gauge and sampling port which were metal) shown inFigure 1. Helium (He) was bubbled at 350 ml min-' through thebuffer solution after passing through a flow meter (Matheson603 flow tube). The He purge gas stream then passed sequentiallythrough trap A (held at -80C to remove the bulk of the watervapor), trap B (held at -196°C to collect the volatile N com-pounds), a column containing glass beads coated with sulfuricacid-dichromate solid oxidizer (preoxidizer) as described byKlepper (5) and Harper (4), and finally through a fritted glassdispersion tube into 20 ml of trapping solution (7.5 g tartaricacid, 0.75 g sulfanilamide, 0.025 g n-1-naphthylethylene-diaminediHCl and 0.025 g disodium 2-naphthol-3,6-disulfonate per literwater). The preoxidizer column oxidized any NO that was notcompletely collected in trap B to NO2 which is completely solublein the trapping solution (2 NO2 + H20 -- NO2- + NO3- + 2H'). It is known that under the trapping conditions used thereaction is not stoichiometric and favors NO- formation overNO3- formation. The exact ratio is, however, unknown. TheNO2- reacts with the Greiss-Saltzman reagents to form an azochromophore which was measured spectrophotometrically. Thisallowed estimation of the amount of NO or other organic Ncontaining compounds that were not totally collected in trap B.The buffer and leaf strips were purged with He for 2 h (the first15 min without liquid N2 around cooling trap B in order topurge the system of air). At the end of this time, stopcocks 1 and2 (Fig. 1) were closed to isolate trap B (preliminary work revealedthat only trace amounts of NO and N20 were collected in trap
A). Trap B was allowed to warm to room temperature (about 1h) and the concurrent increase in pressure was monitored (pres-sure gauge, Fig. 1). Before withdrawing samples from trap B,syringes and needles were thoroughly purged with He. A 0.5 mlaliquot was withdrawn from the sampling port (Fig. 1) andinjected directly into a Hewlett-Packard model 5890A GC fittedwith a thermal conductivity detector (TCD) and a 100/120 meshporopak N column (6.02 m x 2 mm i.d. stainless steel). Theoven temperature was programmed to hold an initial tempera-ture of 60°C for 3 min and then increase 20°C/min to 120°Cwhere the temperature was held. The carrier gas was He at a flowrate of 45 ml min-'. Peaks were quantitated using a Nelsonanalytical 3000 series chromatography data system (Nelson an-alytical, Inc., Cupertino, CA) linked to an IBM PC for datareduction. Retention times and peaks obtained were comparedand calibrated with authentic standards of NO (cp grade, M.G.Burdett Gas Products Co., North Branch, NJ), N20 (AltechAssoc., Inc., Deerfield, IL), air and CO2 (Union Carbide Corp.,Linde Div., New York, NY).GC Analysis of Volatile Organic Carbon Compounds. Organic
carbon compounds co-trapped with NO, N20, CO2, and air intrap B during the purged in vivo NR assay of soybean leaf sliceswere analyzed by GC as described except that: (a) a flameionization detector (FID) was used, (b) the column was 1.5 mlong, (c) the oven was operated isothermally at 170°C, and (d)N2 at a flow rate of 35 ml min-' was the carrier gas. Theseoperating conditions provided greater sensitivity in the detectionof methanol, acetaldehyde, ethanol, and acetaldehyde oxime.The identities of peaks obtained were confirmed by GC/MS (seebelow) and by comparing GC retention times ofunknowns withauthentic standards of methanol (E. M. Industries, Inc., Gibbs-town, NJ), acetaldehyde (Aldrich Chem. Co., Milwaukee, WI),ethanol (USI Chem. Co. Tuscola, IL.), and acetaldehyde oxime(Sigma Chem. Co., St. Louis, MO).GC/MS Identification of Leaf Volatiles from Soybean. A Hew-
lett-Packard model 5985 GC/MS system was used to confirmthe identity of the compounds evolved from soybean leaf slicesand retained in trap B (Fig. 1) during the purged in vivo NRassay. NR assay conditions used to generate '5NO and '5N20were as described except that 10 mM KNO3 was enriched with86.9, 10.1, or 0.366 atom % '5N. Aliquots from trap B wereinjected into the GC and the effluent from the column wasallowed to flow directly into the MS. The GC operating condi-tions and column were exactly as described above, depending onthe sample of interest. The mass spectrometer was operated inthe electron impact mode. The electron energy equaled 70 eVand the source temperature was 200°C.
Species NO(X) Evolution. NO(X) evolution rates during the invivo NR assay of several legume species were tested as previouslydescribed (4), except that propanol was omitted from the NRassay buffer. Nitrite accumulation in the incubation buffer wasdetermined as described (13).
FIG. 1. Diagram of the system used for cryogenictrapping of NO and N20 produced during the He-purged in vivo NR assay of soybean and winged beanleaflets.
Water Both Trap A TrapB(30°C) Dry Ice/Aceone Liqid N
Table I. Amount ofAcetaldehyde Oxime, NO, or NO(X)from SoybeanLeafDiscs Detected as NO2- by the Preoxidizer/Greiss-Saltzman
Method in the Absence or Presence ofa Water TrapNO(x) from soybean leaf discs was assayed by placing 250 mg of leaf
discs in a buffer solution (10 ml) containing 0.1 M K2HPO4-KH2PO4 (pH7.5), and 0.05 M KNO3. The buffer solution was purged with N2 at aflow rate of 150 ml min-' for 30 min. Acetaldehyde oxime was assayedin the same manner by replacing the KNO3 and leaf discs with 10 mMacetaldehyde oxime. Nitric oxide was assayed by injecting an aliquot ofNO directly into the purge gas stream. The 20 ml water trap was placedbetween the purged buffer solution or point of injection of NO and thepreoxidizer/Greiss-Saltzman reagents.
Acetaldehyde Oxime Removal by Water. Table I shows theamount of NO(X), NO, or acetaldehyde oxime recovered as N02(preoxidizer/Greiss-Saltzman assay) from soybean, from a 10mM solution of acetaldehyde oxime or from authentic NO,respectively, when the N2 purge gas stream was or was not passedthrough a water trap. The presence or absence of the water trapdid not appear to affect the amount of NO(.) recovered as N02from soybean. Also, the water trap did not remove authentic NOfrom the purge gas stream. However, the water trap removed96% of the acetaldehyde oxime from the purge gas stream sothat only trace amounts were detected by the Greiss-Saltzmanreagents.GC Analysis of Volatiles Collected in Trap B. Observing trap
B after the 2 h purged in vivo NR assay of soybean leafletsrevealed that a compound resembling bluish-white 'snow' hadbeen collected. The compound was trapped in a concentratedband on the interior glass tube of trap B at a level just above theliquid N2 that was used to cool the trap. The snow turned to ablue fluid upon warming, and at room temperature the trapcontained a gas that had a brown tint (volatiles collected fromwinged bean had the same characteristics). It is known that NOforms a bluish-white solid, a blue liquid, and a colorless gas,depending on temperature. Furthermore, NO is known to reactrapidly with 02 to form NO2 (2 NO + 02 -- 2 NO2) which is abrown gas. Although the assay was done under anaerobic con-ditions, small amounts of02 (bp -1 82.96°C) in trap B (- 196°C)could have occurred from impurities in the He purge gas used
or from dissolved 02 in the incubation buffer that was notcompletely removed during the 15 min purge before trap B wascooled. Withdrawing a sample of gas from trap B and injectingit directly into the Greiss-Saltzman reagents revealed that colorformation occurred, likely from N02, but that the amount ofN02 present was minimal (Table II). Attempts were not madeto detect N02 by the GC method employed due to its lowconcentration and its high reactivity with the Poropak columnused in the GC analysis separation (16).Chromatogram tracings using a TCD during GC analysis of
volatiles collected in trap B when either winged bean, soybean,or the soybean nr, mutant were used in the assay are shown inFigure 2. Peak number 1 is an air contaminant peak that wasobserved even in blank injections of He and could never betotally eliminated. Peaks 2, 3, and 4 had the exact retentiontimes as authentic NO, C02, and N20, respectively. Quantitationof these peaks revealed that winged bean evolved greater quan-tities ofNO than soybean (Fig. 2, A and B), and in both speciesNO production was greater than N20 production (Table II). Theamount of N compounds passing through trap B and detectedby the preoxidizer/Greiss-Saltzman reagents was less than 3% ofthe total amount ofNO collected in trap B, and the amount ofN02 believed to be formed from the reaction ofNO +02 in trapB was less than 3% ofthe total amount ofNO collected. The GCtrace from the nr, mutant lacked both the NO and N20 peak(Fig. 2, C and CO).When the FID was used in GC analysis, four additional
compounds were found to be evolved from soybean leaf strips(Fig. 3). Peaks 1, 2, and 3 had retention times that matchedmethanol, acetaldehyde, and ethanol standards, respectively. Theidentity of these peaks was confirmed by GC/MS (data notshown). The identity of peak 4 is unknown. These four peakswere not quantitated but they appeared to be evolved in onlytrace amounts during the assay. A peak corresponding to theacetaldehyde oxime standard was not obtained from the soybeansample (Fig. 3). It was necessary to use two detection systemssince the TCD was not sensitive enough to detect the low levelsofmethanol, acetaldehyde, and ethanol evolved during the assay,and the FID did not detect NO, N20, C02, or air.GC/MS Identification of "5NO, '5N20, C02, and Air. The mass
spectrum of peak (identified as air in Fig. 2) from soybeanrevealed an intense peak at m/z 28 and minor peaks at m/z 14,16, and 32 (corresponding to '4N'4N+, '4N+, 160+ and 160160+,respectively; Fig. 4). The mass spectrum of peak 2 (identified asNO in Fig. 2) from soybean when 0.366 atom % '3N-N03- wasused in the assay medium (Fig. 5A), revealed an intense peak atm/z 30 (corresponding to '4NO+) along with minor peaks atm/z 28 and 14 ('4N'4N+ and '4N+, respectively). When 10.1%'5N-NO3- was used (Fig. SB) peaks at m/z 14, 30, and 31 ('4N+'4NO+, and "5NO+, respectively) were seen with the m/z 31 peak
Table II. Amounts ofNO, N20, and NO2 Detected in Trap B, and Gaseous N Compound(s) that Passedthrough Trap B during the in Vivo NR Assay ofSoybean and Winged Bean Leaflets
The assay was performed as described in Figure 2 legend. NO and N20 were quantified by GC analysisusing a TCD. NO2 was quantified by withdrawing a 0.5 ml aliquot from trap B, injecting it directly into theGreiss-Saltzman reagents, and measuring N02- formed. The N compound(s) passing through trap B passedthrough the preoxidizer and were quantified as NO2- in the Greiss-Saltzman reagents (Fig. 1).
NCompound(s)Plant Material NO N20 NO2 Passing through
FIG. 2. Chromatogram tracings from GC analysis, usingthe TCD system, of compounds collected in trap B whenleaf tissue either from (A) winged bean, (B) Williams soy-
bean, or (C) the nr, mutant soybean was used in the He-purged in vivo NR assay. Al, B,, and C, show magnifiedtracings of the 5 to 6 min retention time period of A, B,and C, respectively. The NR assay medium consisted of 15g of leaf slices and 500 ml of buffer containing 0.1 M
K2HPO4-KH2PO4 (pH 7.5) and 0.05 M KNO3. The heliumflow rate was 350 ml min-'. Trap B was not cooled for thefirst 15 min of the assay in order to purge most of the airfrom the system.
Retention Time (min)
2
.!- o-
._
0
i 2 3 4 5 6 7 9
Rtntion Time (min)FIG. 3. Chromatogram tracings from GC analysis. using the FID
system, of compounds evolved from the He-purged in vivo NR assay ofsoybean and collected in trap B, compared with selected authenticstandards. See Figure 2 legend for experimental details.
tI"a
el a
2& 4
30m/z
40 50
FIG. 4. Mass spectrum from GC/MS analysis of peak I (air; Fig. 2).Peaks 14, 16, 28, and 32 correspond to the ions '4N+, 160+, '4N'4N+, and160160+, respectively. The assay medium contained 10 mm KNO3 en-
riched with either 0.366, 10. 1, or 86.9 atom % '5N; however, the spectrumwas the same at all atom % '5N concentrations used. This peak isconsidered a contaminant since it was seen in the same quantity duringblank injections. See Figure 2 legend for experimental details.
o00s 30
K A 0.366%J-N0
40-
20- 14loc ... ~~30go B 10.1 % trN0o
20 140 .,.,...............................
100-15 31
8o' C 86.9 % N-NO;3
40 _
20,.,.-- ,;5-lo0 20 30 40 50
mAFIG. 5. Mass spectrum from GC/MS analysis of peak 2 (NO; Fig. 2)
when either (A) 0.366, (B) 10. 1, or (C) 86.9 atom % '5N-NO3; (10 mM)was included in the assay medium during the He-purged in vivo NRassay of soybean leaflets. The peaks at 14, 28, and 30 in (A) correspondto the ions '4N+, '4N'4N+, and 14NO', respectively. The peaks at 14, 30,and 31 in (B) correspond to the ions 14N+, 14NO+, and "5NO+, respectively.The peaks at 14, 30, and 31 in (C) correspond to the same ions as in (B),and the peak at 15 corresponds to '5N+. See Figure 2 legend for experi-mental details.
being about 11% of the sum of the m/z 30 and 31 peaks. Using86.9 atom % '5N-NO3 (Fig. 5C), peaks at m/z 14, 15, 30, and31 were seen ('4N+, '5N+, '4NO+, '5NO+, respectively). However,at the highest level of enrichment the m/z 31 peak was 81% ofthe sum of the m/z 30 plus 31 peaks. The mass spectrum of peak3 (identified as CO2 in Fig. 2) revealed a very intense peak atm/z 44 ('2C'60'60+) and minor peaks at m/z 28 and 16 (corre-
sponding to '2C'6O+ and 160+, respectively; Fig. 6). The massspectra of peaks (air) and 3 (CO2) did not change with thechange in '"N-NO3 enrichment. The mass spectrum of peak 4(identified as N20 in Fig. 2) from the same three "5N experimentswhen 0.366 atom % '5N-NO3- was used in the assay medium(Fig. 7A) revealed an intense peak at m/z 44 and smaller peaksat m/z 30 and 28 (corresponding to '4N'4NO' and the fragments'4NO+ and '4N'4N+, respectively). With 10.1 atom % '5N-NO3-(Fig. 7B), peaks with m/z 46, 45, 44, 31, 30, and 14 were observed("5N`NO+, 15N'4NO+, '4N'4NO+, and the fragments '5NO+,'4NO+, and '4WN, respectively). Assuming that the '4N and 'INin N20 follow the binomial distribution (x + y) = xn + 2xy +y', and there is no contribution from isotopes of 160+ ('7O+ and180+), then N20 derived from atom % '"N-NO3- shouldtheoretically contain 80.8% '4N'4NO+, 18.2% '"N'4NO+, and1.0% I"N''NO+. The actual values obtained from Figure 7B were
1.8cc
i20
10 2D 30MAz
40 50
FIG. 6. Mass spectrum from GC/MS analysis of peak 3 (CO2; Fig. 2).Peaks 16, 28, and 44 correspond to the ions 160+, 12C'6O+, and12C'6O'6O+, respectively. The assay medium contained 10 mM KNO3enriched with either 0.366, 10.1, or 86.9 atom % '"N; however, thespectrum was the same at all atom % 'IN concentrations used. See Figure2 legend for experimental details.
10
a
6
4
2
c0
a)
.2 2
0-
'is
4
10 2a 30m/z
40 50
FIG. 7. Mass spectrum from GC/MS analysis of peak 4 (N20; Fig. 2)when either (A) 0.366, (B) 10.1, or (C) 86.9 atom % "IN-NO3- (10 mM)was included in the assay medium during the He-purged in vivo NRassay of soybean leaflets. The peaks at 28, 30, and 44 in (A) correspondto the ions 14N14N+, 14NO+, and 14N14NO+, respectively. The peaks at 14,30, 31, 44, 45, and 46 in (B) correspond to the ions 14N', 14NO+, "5NO+,14N'14NO+, 15N'4NO+, and 15"N"NO+, respectively. The peaks at 30, 31,44, 45, and 46 in (C) correspond to the same ions as in (B), and the peakat 15 corresponds to "N+. See Figure 2 legend for further experimentaldetails.
78, 19, and 3%, respectively. When 86.9 atom % "5N-N03- wasused (Fig. 7C), the same peaks were observed as when the 10.1%1N03- was used except a m/z 15 peak corresponding to a 15N+fragment replaced a m/z 14 peak corresponding to a '4N+ frag-ment. Using the same assumptions as above N20 derived from86.9 atom % '5N-NO3 should theoretically contain 1.7%14N14NO+, 22.8% 15N'4NO+, and 75.5% '5N'5NO+. The actualvalues obtained from Figure 7C were 4.5, 22.4, and 73.1%,respectively.
Species NO(.) Evolution. Winged bean evolved the greatestquantity of NO(X) of any species tested (Table III). All of theGlycine sp. tested, except the nr, mutant, evolved significantquantities of NO(X), while N. wightii, P. montana, and P. thun-bergiana evolved low levels of NO(X). These are the only speciesto date that are known to evolve NO(x). At least 30 other speciestested did not evolve NO(X) (SA Ryan, personal communication).
DISCUSSION
Significant quantities of both NO and N20 were determinedby GC and GC/MS to be evolved during the purged in vivo NRassay ofsoybean leaflets (Table II). The estimation ofthe amountof NO(x) evolved by soybean and winged bean differed whenquantitation was done by GC (Table II) or the preoxidizer/Greiss-Saltzman method (Table III). This is expected due todifferences in bubbler design, buffer volume, gas flow rate, andlength of assay time which all affect the rate of NO(x) evolution.Since N20 is not detected by the preoxidizer/Greiss-Saltzmanassay and no other N compounds were detected by GC, NOmust be responsible for the results seen by Harper (4). Thesepresent results agree with the findings of Harper (4) and Klepper(5) that soybean leaves evolve significant quantities of NO(X).Both Klepper (5) and Harper (4) suspected that greater quantities
Table III. NR Activity and Concurrent NO(x) Evolution ofSeveralLegume Species during the Purged in Vivo NR Assay
The assay mix (10 ml) consisted of 0.1 M K2HPO4-KH2P04 (pH 7.5),0.05 M KNO3, and 250 mg of leaf discs. N2 was the purge gas at 150 mlmin-'. The assay time was 30 min.
of NO than NO2 were being evolved, which was also observedin the present study. In fact, the NO2 found in trap B wasattributed to the reaction ofNO with contaminating levels of 02.If NO2 itself was produced, it would be hard to imagine it beingpurged from the incubation buffer based on its complete solu-bility in water (4, 5). Further, NO2 (bp 21.2°C) would have beenpredominately trapped in trap A (-80°C), and analysis of thewater vapor in trap A by the Greiss-Saltzman reagents did notindicate the presence ofNO2. The '5N data confirmed the identityof NO and N20, and proved that both "5NO and '5N20 wereproduced from '5N-NO3 during nitrate reduction. Althoughacetaldehyde oxime was previously reported to be the major Ncompound evolved from soybean leaves during the in vivo NRassay (10), this product was not detected by our methods. Theobservation that authentic acetaldehyde oxime can be removedfrom the purge gas stream with a water trap, whereas neither theproduct evolved from soybean leaf slices nor authentic NO(Table I) can be removed by a water trap, gave us a preliminaryindication that the product evolved was not acetaldehyde oxime.It is unknown why a previous study using cryogenic trappingand MS analysis did not detect NO (10). It is possible that thehighly reactive NO could have combined with acetaldehyde orone of the other organic carbon compounds found in trap B,generating acetaldehyde oxime during the week long incubationperiod used to allow isomerization (10). The evolution of N20had been previously reported (10).With the exception of the nr, mutant, all the Glycine sp.
examined evolved NO(X). The other species that evolved NO(X),Neonotonia wightii, Pueraria montana, Pueraria thunbergiana,and Psophocarpus tetragonolobus, are classified with the Glycinesp. in the same subfamily (Papilionoideae) and tribe (Phaseoleae)of the family Leguminosae (1). N. wightii occurs in the samesubtribe (Glycininae) as the Glycine sp. and was at one timeclassified as a Glycine sp. until given the new generic name byLackey (7). The NO(x) evolution data (Table III) indicate that N.wightii and the Glycine sp. are dissimilar. Whether Pueraria sp.are in the subtribe Diocleinae, or Glycininae is not clear (6).Lackey (6, 7) has suggested that a revision of the genus Puerariais needed and that upon this revision a few Pueraria sp. may bereferred to the genus Neonotonia. Winged bean (subtribe Phas-eolinae) is the most distant relative of the species listed in TableIII, yet it has the highest rates of NO(x) evolution of any speciestested; the significance of this is unknown.The nr, mutant did not evolve NO(x) (Table III) and also has
lower NR activity (due to a loss of the constitutive NR enzymes)than the wild-type soybean. However, exogenous NO2- suppliedto the nr, mutant still does not invoke NO(x) evolution. Therefore,the lack of substrate cannot be the cause of the loss of NO(x)evolution (1 1, 14). There will undoubtedly be other species foundthat evolve NO(x), but it is possible that these species may berestricted to the legume family since several species tested frommany diverse families did not exhibit NO(x) evolution. Additionalresearch is needed before the exact phylogenetic significance ofNO(x) evolution is known.The pathway of NO3- reduction during microbial denitrifica-
tion is generally thought to be: N03- NO2--* NO -- N20 --
N2. However, there still remains controversy as to the role ofNOand N20 as intermediates (3). It has been shown by GC/MS thatboth NO and N20 are produced from a Cyt oxidase (nitritereductase) isolated from Pseudomonas aeruginosa (17). A Cyt coxidase isolated from beef heart has the ability to catalyze thereduction of NO to N20 and the reversible oxidation ofNO toN02- (2). A Cyt cd from Alcaligenesfaecalis is known to catalyzethe reduction of N02- to NO and the reduction of NO to N20(8, 9). It is interesting that the nrl mutant which does not evolveNO or N20, has decreased Cyt c reductase activity which isassociated with the loss of the constitutive NR activity (12).Whether or not the Cyt c reductase can act as a Cyt oxidase(nitrite reductase) is unknown. Recently, two forms of the con-stitutive NR enzyme (c1NR and c2NR) have been purified fromwild-type soybean leaflets (15) and we are currently investigatingifone or both ofthe constitutive NR enzymes or another enzymeis responsible for the NO and N20 production.
Acknowledgments-The authors thank J. C. Nicholas and D. J. Hendren fortechnical assistance, and Drs. R. H. Hageman and R. J. Volk for helpful commentson this manuscript. Winged bean seed was generously provided by the Niftalproject, University of Hawaii.
LITERATURE CITED
1. ALLEN ON, EK ALLEN 1981 The Leguminosae: A Source Book of Character-istics, Uses, and Nodulation. University of Wisconsin Press, Madison
2. BRUDVIG GW, TH STEVENS, SI CHAN 1980 Reactions of nitric oxide withcytochrome c oxidase. Biochemistry 19: 5275-5285
3. FIRESTONE MK 1982 Biological denitrification. Agronomy 22: 289-3364. HARPER JE 1981 Evolution of nitrogen oxide(s) during in vivo nitrate reductase
assay of soybean leaves. Plant Physiol 68: 1488-14935. KLEPPER L 1979 Nitric oxide (NO) and nitrogen dioxide (NO2) emissions from
herbicide-treated soybean plants. Atmos Environ 13: 537-5426. LACKEY JA 1977 A revised classification ofthe tribe Phaseoleae (Leguminosae:
Papilionoideae), and its relation to canavanine distribution. Bot J Linn Soc74: 163-178
7. LACKEY JA 1977 Neonotonia, a new generic name to include Glycine wighii(Amott) Verdcourt (Leguminosae; Papilionoideae). Phytologia 37: 209-212
8. MATSUBARA T, H IWASAKI 1971 Enzymatic steps of dissimilatory nitritereduction in Alcaligenesfaecalis. J Biochem 69: 859-868
9. MATSUBARA T, H IWASAKI 1972 Nitric oxide-reducing activity of Alcaligenesfaecalis cytochrome cd. J Biochem 72: 57-64
10. MULVANEY CS, RH HAGEMAN 1984 Acetaldehyde oxime, a product formedduring the in vivo nitrate reductase assay of soybean leaves. Plant Physiol76: 118-124
1 1. NELSON RS, SA RYAN, JE HARPER 1983 Soybean mutants lacking constitutivenitrate reductase activity. I. Selection and initial plant characterization. PlantPhysiol 72: 503-509
12. NELSON RS, L STREIT, JE HARPER 1984 Biochemical characterization ofnitrateand nitrite reduction in the wild-type and a nitrate reductase mutant ofsoybean. Physiol Plant 61: 384-390
13. NICHOLAS JC, JE HARPER, RH HAGEMAN 1976 Nitrate reductase activity insoybeans (Glycine max [L.] Merr.). I. Effects of light and temperature. PlantPhysiol 58: 731-735
14. RYAN SA, RS NELSON, JE HARPER 1983 Soybean mutants lacking constitutivenitrate reductase activity. II. Nitrogen assimilation, chlorate resistance, andinheritance. Plant Physiol 72: 510-514
15. STREIT L, RS NELSON, JE HARPER 1985 Nitrate reductases from wild-type andnr,-mutant soybean (Glycine max [L.] Merr.) leaves. I. Purification, kinetics,and physical properties. Plant Physiol 78: 80-84
17. WHARTON DC, ST WEINTRAUB 1980 Identification of nitric oxide and nitrousoxide as products of nitrite reduction by Pseudomonas cytochrome oxidase(nitrite reductase). Biochem Biophys Res Commun 97: 236-242
