Plant Physiol. (1973) 51, 332-336

In Vivo Assay of Nitrate Reductase in Cotton Leaf Discs

EFFECT OF OXYGEN AND AMMONIUM'

Received for publication August 11, 1972 J. W. RADIN Western Cotton Research Laboratory, United States Department ofAgriculture, 4135 East Broadway, Phoenix, Arizona 85040

ABSTRACT was quite uncritical between 0.3 and 2%; within this range of concentrations, there was no difference in the response to Factors affecting nitrate reduction by leaf discs of cotton nitrate. In some studies disodium arsenate or other salts were (Gossypium hirsutum L.) were investigated. When incubated in added to the medium as experimental treatments. Generally 30 mM nitrate, discs reduced nitrate much more slowly under air there were 3 discs per tube, with three replicates per treat- or 02 than under N2. Inhibition by 02 did not occur at nitrate levels of 100 mM or greater. Treatment with arsenate had little ment. For time course studies, 20 ml of assay medium and 20 effect under Na but stimulated nitrate reduction under air. discs were placed in 50-ml flasks, with two replicates per treat- Similarly, ammonium inhibited nitrate reduction, with the in- ment. Tubes or flasks were connected to a manifold and evac- hibition being partially relieved by arsenate. Uptake of nitrate uated with a vacuum pump to a pressure of less than 5 mm was unaffected by ammonium. The NAD/NADH ratio increased Hg. Anaerobiosis was maintained while releasing the vacuum in response to both oxygen and amnmonium. The effects of these by introducing nitrogen gas from a cylinder connected to the treatments on nitrate reduction can be explained by competition line. The procedure was repeated once, and the vessels were with nitrate for NADH generated by glycolysis. removed from the line and quickly stoppered. During infil- tration the discs became wetted and sank to the bottom of the medium. Infiltration was carried out in an ice bath. After infiltration, the samples were placed in a water bath at 30 C in darkness and agitated during incubation. At the end of a run they were transferred to an ice bath, and aliquots of the medium were removed for analysis. (For time course studies, the flasks were stoppered with serum caps and 0.2-ml Several investigators have described an in vivo assay sys- aliquots were removed during the incubation.) Nitrite was tem for the reduction of nitrate to nitrite (3, 7, 9-11, 19). determined by the procedure of Kende et al. (10). There was This system depends upon the absorption of nitrate, endoge- considerable variation between experiments, probably arising nous generation of NADH, and subsequent release of nitrite from differences in source material. However, a single large into the medium. In addition, accumulation or reduction of leaf usually produced sufficient discs for any one experiment, nitrite by the tissue can diminish the amount secreted into and replicates were thus usually within 5% of the treatment the medium (4, 11). Thus, the apparent activity of NRW (rate means. of appearance of nitrite) depends upon several physiological In several studies the oxygen concentration was altered by and biochemical processes. This report describes the effects releasing the vacuum with air instead of nitrogen, or by bub- of various incubation conditions and metabolites on the ap- bling air or pure oxygen through the media for 30 sec after parent activity of NR. infiltration. In the latter procedure the control samples were given nitrogen for 30 sec. In experiments without vacuum in- MATERIALS AND METHODS filtration, oxygen, air, or nitrogen was bubbled through the samples for 1 min to establish the proper atmosphere. Plant Material. Seeds of cotton (Gossypium hirsutum L. cv. Nitrate Uptake. Twenty leaf discs were infiltrated in flasks Deltapine 16) were germinated in a greenhouse in pots con- containing 10 ml of 1 mM KNO2, and the flasks were in- taining a mixture of sand and peat moss and watered with a cubated anaerobically in darkness at 30 C. Loss of nitrate modified Hoagland's solution (0.4 strength). Discs 10 mm in from the medium was followed with a nitrate ion electrode diameter were cut with a cork borer from healthy, fully ex- (Orion Research Inc.).' Each treatment was replicated three panded leaves, taking care to exclude the major veins. times. This low nitrate concentration was used to enable Assay Procedure. The assay medium of 1 % propanol (v/v), measurable differences to develop between initial and final 0.1 M phosphate buffer, pH 7.5, and the appropriate amount concentrations. The electrode's selectivity precluded any in- of potassium nitrate or nitrite in a total volume of 3 ml, was terference from nitrite ions. put into 18- X 150-mm test tubes. Propanol concentration NAD(H) Detvrminations. Nucleotides were extracted from lyophilized leaf tissues bv the procedures of Guinn (unpub- I Contribution of the Agricultural Research Service, United States Department of Agriculture, in cooperation with the Arizona sMention of a trademark or proprietary product does not con- Agricultural Experiment Station. Journal Paper 1929 of the Arizona stitute a guarantee or warranty of the product by the U. S. De- Agricultural Experiment Station. partment of Agriculture, and it does not imply approval to the 2Abbreviations: NR: nitrite reductase, NiR: nitrite reductase. exclusion of other products that may also be suitable. 332 Plant Physiol. Vol. 51, 1973 IN VIVO NITRATE REDUCTION IN COTTON 333 lished) modified from et al. The Ben-Hayyim (2). techniques I - for NAD (NADH) included extraction with ice-cold 0.1 N 6 H2S04 (0.25 N NaOH), incubation at 30 C for 30 min, adjust- I ment of the pH to 3.0 (7.6), and passage through a 5-cm 2 column of Polyclar AT (Sigma). The pH of both eluates was adjusted to 8.7 with tris for assay by the method of Yamamoto /11 0 (20). Alcohol dehydrogenase and NADH diaphorase were O/ purchased from Internal standards were used to cor- x 4 Sigma. /// AIR 0 rect for inhibition. I0 0 RESULTS Effect of Oxygen on Nitrate Reduction. Appearance of ni- trite in the incubation medium was clearly enhanced under I I I I __j anaerobic conditions (Table I). When tissues were air or given 0 0.2 0.4 oxygen either with or without vacuum infiltration, then in- NO3 CONCENTRATION, MOLAR cubated in 30 mm nitrate, the rate of appearance of nitrite was substantially diminished. In all cases, infiltration ap- FIG. 1. Effect of nitrate concentration on apparent NR activity proximately quadrupled the rate, probably because of greater under aerobic and anaerobic conditions. Treatments were effected nitrate uptake. In addition, infiltration eliminated a 15-min by releasing the infiltration vacuum with either air or N2. These lag phase, possibly by removing all air from intercellular results are typical of several experiments. spaces (data not shown). Vacuum infiltration was routinely used in the other here. experiments reported Effect of Oxygen on Nitrite Reduction. Klepper et al. (11) Inhibition by oxygen could be observed only at external nitrate concentrations less than 100 mm observed the reduction of nitrite by tomato leaf discs incu- (Fig. 1). Strictly bated in darkness, although it occurred much more anaerobic conditions released with re- slowly (vacuum nitrogen) than in the light. Dark reduction of nitrite obviously could sulted in a fairly sharp peak of activity at 30 to 60 mm ni- 100 cause errors in the estimation of NR activity by in vivo tech- trate; at levels of mm and greater, nitrate affected activity niques. In the present nitrite very little. When air, rather than was introduced experiments, reduction could not nitrogen, be measured directly, as cotton leaf discs secreted to release the vacuum, then maximal was found at nitrite into activity the medium even when incubated with nitrite and without 100 mM nitrate. This was smaller and broader peak than the nitrate (Table II). Nevertheless, indirect procedures could be anaerobic peak; the with nitrate was however, plateau high only used. When leaf tissue was treated for 20 hr in darkness with slightly lower with air than with nitrogen. This slight differ- distilled water, most of the ability to reduce nitrate was lost ence between treatments with high nitrate may explain why (Table II). However, aged discs other investigators (7, 11), who used 200 mm nitrate, found demonstrated net uptake of strict anaerobiosis nitrite when incubated with this ion. Under these conditions unnecessary. one Several of the effect can be would expect loss of NR activity and retention of NiR explanations oxygen advanced. activity (1). First, nitrate might be taken up in greater amounts under Thus, the appearance of the ability to take up nitrogen than under air. This is since ion in nitrite suggests a constant activity of NiR, superimposed upon unlikely, uptake a darkness is upon oxidative pattern of declining NR activity (Table II). That the move- generally dependent phosphoryla- ment of nitrite reflected tion (5). Second, a product of aerobic or anaerobic metabo- enzyme activities in aged discs was confirmed lism may directly change the affinity of NR for its substrate. by the complete absence of accumulated nitrite This possibility cannot be adequately tested without exten- in discs incubated with either nitrate or nitrite. Thus, aged sively purified NR for kinetic studies. leaf discs could reduce nitrite at a measurable rate, even Third, oxygen may incubated cause depletion of available NADH for nitrate reduction. though in darkness. Since NiR is relatively stable This suggestion was made by Ferrari and Varner (3) and is supported by the findings of Klepper et al. (11) that genera- Table II. Inzcreases or Decreases in Nitrite of Incubation Media tion of NADH, rather than NR itself, can limit in vivo nitrate Caused by Freshly Cut or Aged Discs reduction. oxygen may stimulate Fourth, nitrite reduction by Aged discs were pretreated for 20 hr with distilled water in NiR. This last possibility was examined further. darkness. Assays were performed under N2 with either 30 mm nitrate or 100lM nitrite in the incubation medium. A negative rate Table I. Rate of Appearance of Nitrite in the Incubation Medium indicates a net decrease in nitrite. Values given with standard with Various Treatments errors of the means. In samples which were vacuum-infiltrated, N2, air, or O2 was bubbled through the medium for 30 sec. In samples not infiltrated, Nitrite Released the gas was bubbled for 1 min. All media contained 30 mm KNO,. Incubation Medium Values are given with standard errors of the means. Fresh Aged

Nitrite Released pmoles N022/gl - kr-l Treatment A. Control (no nitrate 1.6 i 0.3 0.4 0.1 Infiltrated Not infiltrated or nitrite) B. Nitrate 7.8 : 0.1 1.2 ± 0.1 umoles N02-/g-1-hr- C. Nitrite 0.5 0.2 -1.0 4 <0.1 N2 7.8 i 0.1 1.8 0.1 Air 5.4 i 0.3 1.4 0.1 Total nitrite reduced 1.1 i 0.4 1.4 i 0.1 02 3.1 + 0.2 0.9 0.2 (A-C) 334 RADIN Plant Physiol. Vol. 51, 1973 the latter proportional to nitrite concentration (Fig. 2). Thus, 2 I dC,'dt = A - BC 0 and B are constants, and Li) where C is nitrite concentration, A

- t is time. This equation when integrated yields (IE0 I C = (AIB) (1 - eBl) From the exponential nature of this equation, it can be de- LUJ AIR - duced that the deviation from linearity would be minimized -j(N by a short assay time. For a typical 1-hr incubation under anaerobic conditions, the error from nitrite reduction was 0 0 only about 5 %. z I I 0 40 80 Effect of Ammonium on Nitrate Reduction. Leaf discs given 20 meq/liter (NH,)2SO4 typically showed a 20 to 30% NO2 CONCENTRATION, PMOLAR decrease in nitrate reduction (Fig. 3). The decrease was ap- FIG. 2. Effect of nitrite in the incubation medium on the rate parent as early as 30 min after the start of incubation. Simi- of nitrite production under aerobic and anaerobic conditions. Discs lar decreases were observed for equivalent concentrations of were cut from fresh leaf material. Nitrate was not present in the NH4Cl and NH4NO3 (the latter with the KNO3 concentration medium. Treatments were effected by releasing the infiltration adjusted appropriately), but K2S04 and KCI had no effect. vacuum with either air or N2. These results are typical of several Therefore, the decrease appeared to be specific for the am- experiments. monium ion. Several investigators have shown a decrease in nitrate up- take by plants given ammonium (13, 16). In cotton leaf discs 30 incubated in 1 mM KNO3, there was no effect of ammonium CONTRO L on nitrate uptake (Table III). This result supports a similar observation by Smith and Thompson (18) in barley roots. It is 70 Li) possible that ammonium affected nitrate uptake differently - 1 the rapidity with 20 100 from mm and 30 mm solutions; however, 0~ "I which nitrate reduction responded to ammonium suggests N H4 could not account for reduced NR ac- + 11 that decreased uptake Li) z tivity (Fig. 3). / , Effect of Arsenate on Nitrate Reduction. Klepper et al. (11) / 10l r/~~~Il/ 4_ 50 z demonstrated a role of glycolysis to supply NADH for nitrate // 0 0 reduction. Thus, any factor decreasing available reducing z >// °.,-. o. INHIBITION I power might be expected to slow nitrate reduction. The

z possibility that oxygen or ammonium acts through this mech- anism was approached both indirectly and directly. In the first o 0 set of experiments, arsenate was added to the incubation me- 0 100 200 300 dium to bypass the ADP phosphorylation step of glycolysis (1 1). Nitrate reduction was increased by arsenate under aerobic TIME, MIN conditions but was slightly inhibited under anaerobic condi- FIG. 3. Time courses for the appearance of nitrite in the incu- tions (Table IV). Similarly, nitrate reduction was stimulated bation medium with and without 20 mM ammonium present. The by arsenate in the presence of ammonium, but there was lit- medium contained 30 mM nitrate. Incubation was strictly anaerobic tle effect in the controls (Table IV). These results are con- (infiltration vacuum released with N2 ). sistent with the hypothesis that the rate of glycolysis, and consequent NADH generation, was limiting under air or in in vivo (1), fresh discs probably also reduced nitrite, but with the presence of ammonium. Presumably reductive amination the process masked by nitrate reduction (Table II). of a'-ketoglutarate was responsible for utilization of reducing Table II shows that nitrite in the medium inhibited the power by ammonium. net release of nitrite by freshly cut leaf discs, probably because Direct Measurement of NAD and NADH. The foregoing of increased nitrite reduction. The inhibition by nitrite was experiments imply that NADH was less available in leaf tis- proportional to concentration under strictly anaerobic condi- sues given either air or ammonium salts. This prediction was tions but was greatly diminished when air was present (Fig. 2). Thus, it is possible that oxygen inhibited nitrite reduction. Table III. Efect of Amnimoniuni oti the Uptake ofNit rate by Cotton With the low concentrations of nitrite and short treatment Leaf Discs periods used, it is unlikely that the response was a manifesta- Discs were incubated in 1 mm. KNO3, and uptake was measured tion of nitrite toxicity. In addition, the lack of effect of nitrite by loss of nitrate from the medium. Rates are given with standard under air also argues against a general toxicity. However, the errors of the means. influence of oxygen was opposite to that frequently observed in nonphotosynthetic tissues (1, 4). Whether oxygen directly Ammonium Concn Nitrate Uptake Rate affected NiR or some other component of the nitrite-reduc- ing system (e.g., ferredoxin) in the leaf discs could not be m31 pnoles/gl-. hr- determined. 0 5.5 +0.2 The rate of change of nitrite concentration with time can be 2 5.1 0.3 expressed as the difference between the rates of nitrate re- 20 5.9 + 0.3 duction and nitrite reduction, with the former constant and Plant Physiol. Vol. 51, 1973 IN VIVO NITRATE REDUCTION IN COTTON .)335 confirmed by extraction and direct measurement of NAD Table V. Pyridine Nucleotide Content and Ratios of Leaf Tissue and NADH in treated tissue. Ammonium increased the Treated with Either Ammonium or Air during Iincubation NAD/NADH ratio from 0.16 to 0.43 (Table V). Even with Tissue was infiltrated with 30 mm nitrate, incubated in darkness ammonium present, the ratios obtained were quite low, since and lyophilized for extraction and analysis. Each value is the the tissue was incubated anaerobically. In leaves incubated in mean of two analyses. The experiment was repeated once, with an aerated medium after infiltration, the NAD/NADH ratio similar results. was increased to 1.01 (Table V). The relative magnitudes of the changes in this ratio are correlated with the degree of in- Treatment NAD NADH NAD/NADH hibition of nitrate reduction (Figs. 1, 3), and also with the effectiveness of arsenate as a stimulant (Table IV). Thus, it is imoles/g dry wi-I likely that both oxygen and ammonium affected nitrate reduc- Control 0.05 0.33 0.15 tion primarily through the availability of reducing power. Ammonium 0.09 0.21 0.43 These results make unlikely the possibility that ammonium Air 0.68 0.67 1.01 or oxygen directly inhibited NR activity. If such were the case, then the decreased rate of nitrate reduction would lead to a decrease in the NAD/NADH ratio. However, turnover of true rate of nitrate reduction in the light cannot be easily esti- NAD and NADH, assumed to occur at a constant rate, was mated from an assay performed in darkness. Unfortunately, not measured. the in vivo assay does not work in the light with green tissue, as nitrite reduction by NiR is greatly increased (11). DISCUSSION Ammonium ions may also compete with nitrate for NADH, Nitrate reduction in the in vivo is probably through reductive amination of a-ketoglutarate. Leech assay normally corre- and Kirk (12), on the basis of differential properties of gluta- lated with extractable NR activity (19), and the assay has mate dehydrogenases in chloroplasts and been used to estimate the potential of a crop to assimilate mitochondria, sug- over a season the gested that reductive amination is specific for NADPH in nitrate growing (7). However, present study higher plants as in microorganisms. However, demonstrates that factors other than NR itself can frequently coenzyme spec- affect the observed rates. Results obtained with the in vivo ificity and relative reaction rates in vitro are highly subject assay, as with the in vitro assay, must be interpreted with to type of buffer and other treatment conditions (14). Others caution. have suggested that the NAD-dependent enzyme catalyzes The three factors investigated as possible sources of error predominantly the reductive reaction (15, 17). In either case, in the assay were nitrite, oxygen, and ammonium. Nitrite under anaerobic conditions the low NAD/NADH ratio (Table buildup in the medium during incubation was found to be of V) would preclude extensive oxidative deamination. Therefore, little concern, as high concentrations of the ion were required the data of the present study cannot be used to decide between to stimulate NiR activity (Fig. 2). On the other hand, oxygen the alternatives. a influence on exerted profound the assay under certain con- Acknowledgments-I am grateful to Dr. Gene Guinn for guidance in the deter- ditions (Table I, Fig. 1). Probably this influence was based mination of nucleotides, and to Dr. Wayne R. Jordan for critical review of the upon competition for NADH (Tables IV, V). Guinn and manuscript. Brinkerhoff (6) showed a large increase in amino acid con- tent of nitrate-fed roots when the nutrient solution became LITERATURE CITED depleted of oxygen. Thus, it can be inferred that the same competition operates in cotton roots. Similarly, Ferrari and 1. BEEVERS, L. AND R. H. HAGEMAN. 1969. Nitrate reduction in higher plants. Varner (3), using aleurone obtained a in- Annu. Rev. Plant Physiol. 20: 495-522. barley layers, great 2. BEN--HAYYIM, G., Z. GROMET-ELHANAN, A-ND M. AvRo.. 1969. A specific and crease in nitrate reduction with either anaerobiosis or inhibi- sensitive method for the determination of NADPH. Anal. Biochem. 28: 6-12. tors of respiratory electron transport. However, in view of 3. FERRARI, T. E. AND J. E. VARNER. 1970. Control of nitrate reductase activity the controversy over the occurrence of mitochondrial respira- in barley aleurone layers. Proc. Nat. Acad. Sci. U.S.A. 65: 729-736. tion in leaves in the light under such conditions the 4. FERRARI, T. E. AND J. E. VARNER. 1971. Intact tissue assay for nitrite reductase (8, 21), in barley aleurone layers. Plant Physiol. 47: 79-794. competition may be lessened or eliminated. Therefore, the 5. FISHER, J. D., D. HANSEN, AN-D T. K. HODGES. 1970. Correlation between ion fluxes and ion-stimulated adenosine triphosphatase activity of plant roots. Table IV. E$Tect of Arsenate oni Appearance of Nitrite from Discs Plant Physiol. 46: 812-814. Inicuibated Either or with 20 m-ni Ammonium in 6. GUIN-N, G. AND L. A. BRIN,ERHOFF. 1970. Effect of root aeration on amino acid Aerobically the levels in cotton plants. Crop Sci. 10: 175-178. Medium 7. HARPER, J. E. AND R. H. HAGEMAN. 1972. Canopy and seasonal profiles of Arsenate was 10 mm. Values given with standard errors of the nitrate reductase in soybeans (Glycine max L. Merr.). Plant Physiol. 49: means. These results are typical of several experiments. 146-154. 8. JACKSON, W. A. AND R. J. VOLK. 1970. Photorespiration. Annu. Rev. Plant Physiol. 21: 385-432. Nitrite Released 9. JAWORSKI, E. G. 1971. Nitrate reductase assay in intact plant tissues. Biochem. Stimulation Treatment by Arsenate Biophys. Res. Comrnmun. 43: 1274-1279. -Arsenate +Arsenate 10. KENDE, H., H. HAsN, .AND S. E. KAYS. 1971. Enhancement of nitrate reduc- tase activity by benzyladenine in Agrostemma githago. Plant Physiol. 48: ,umoles N02-/g'hl-r-I % 702-706. Experiment I 11. KLEPPER, L., D. FLESHER, AND R. H. HAGEMAN. 1971. Generation of reduced nicotinamide adenine dinucleotide for nitrate reduction in green leaves. Control 6.3 +4 0.7 5.7 + 0.3 -10 Plant Physiol. 48: 580-590. Aerated 5.4 i4 0.3 7.6 i 0.1 41 12. LEECH, R. M. AND P. R. KInx. 1968. An NADP-dependent L-glutamate dehydrogenase from chloroplasts of Vicia faba. Biochem. Biophys. Res. Experiment II Commun. 32: 685-690. 13. MINNoTTI, P. L., D. C. WILLIAMS, AND W. A. JACKSON. 1969. The influence Control 7.7 i 0.3 8.2 i 0.3 6 of ammonium on nitrate reduction in wheat. Planta 86: 267-271. Ammonium 4.9 i1 <0.1 6.2 ± 0.3 27 14. PAHLICH, E. AND K. W. Joy. 1971. Glutamate dehydrogenase from pea roots: purification and properties of the enzyme. Can. J. Biochem. 49: 127-138. 336 RADIN Plant Physiol. Vol. 51, 1973

15. PRIESS, J. AND T. KOSUGE. 1970. Regulation of enzyme activ ity in photo- 18. SMITH, F. W. AND J. F. THOMPSON. 1W71. Regulation of nitrate reductase in synthetic systems. Annu. Rev. Plant Physiol. 21: 433-465. excised barley roots. Plant Physiol. 48: 219-223. 16. RAO, K. P. AND D. W. RAINS. 1971. Kinetics of nitrate absorption by barley 19. STREETER, J. G. AND M. E. BOSLER. 1972. Comparison of in vitro and il vivo seedlings. Plant Physiol. 47: S-16. assays for nitrate reductase in soybean leaves. Plant Physiol. 49: 448-450. 17. SINMS, A. P., B. F. FOLKES, AND A. H. BUSSEY. 1968. Mechanisms inxvolved in 20. YAMAMOTO, Y. 1963. Pyridine nucleotide content in the higher plant. Effect of the regulation of nitrogen assimilation in micro-organisms and plants. In: age of tissue. Plant Physiol. 38: 45-54. E. J. Hewitt and C. V. Cutting, eds., Recent Aspects of Nitrogen 'Metabo- 21. ZELITCH, I. 1971. Photosynthesis, Photorespiration, and Plant Productivity. lism in Plants. Academic Press, London, pp. 91-114. Acadeinic Press, New York.