In Vivo Assay of Nitrate Reductase in Cotton Leaf Discs
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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.