MNinimizing Nitrate Reduction during Kjeldahl Digestion of Plant Tissue Extracts and Stem Exudates ' APPLICATION TO 15 N STUDIES

MATERIALS AND METHODS From 10 to 60% of the nitrate present in plant tissue extracts and stem exudates of corn (Zea mays L.) was found to be reduced during Kjeldahl digestion, even in the absence of added reducing agents. This reduction is of particular concern in [15Ninitrate assimilation studies, because it results in an overestimate of nitrate reduction. To overcome this problem, a method was developed for removing nitrate prior to Kjeldahl digestion, thereby preventing nitrate reduction. The procedure utilizes hydrogen peroxide for partial oxidation of organic matter in order to minimi the nitration of organic componds. The free nitrates are then volatized as nitric acid from concentrated sulfuric acid at 950C. When the proposed method was used as a pretreatment to Kjeldahl digestion, less than 0.5% of the appled nitrate was recovered in the reduced nitrogen fracuon of plant tissue extracts and stem exudates. The Kjeldahl method is commonly used in studies of nitrogen assimilation in plants. However, the method must be modified if quantitative results are to be obtained with samples containing nitrate, because some of the nitrate may be reduced during digestion (1). This possibility is of primary concern in the analysis of [15NJnitrate assimilation studies. Any reduction of ['5N]nitrate during Kjeldahl digestion would increase both total nitrogen and atom per cent 15N in the reduced N fraction, thus inflating the apparent assimilation of nitrate. Minimizing nitrate reduction is particularly important in the determination of reduced N in samples that have amounts of nitrate in excess of the amount of reduced N. We have tried both removing nitrate from these aqueous samples with ion exchange resins and reducing the nitrate completely during Kjeldahl digestion, but neither method provided sufficiently accurate results to prepare satisfactory balance sheets for assimilated [15N]nitrate. We report in this paper a volatilization method for removing most of the nitrate from tissue extracts and xylem exudates to preclude the possibility of appreciable nitrate reduction during subsequent Kjeldahl digestion. 'Supported by Grant PCM-7703152 from the National Science Foundation. Paper No. 6804 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh. 2Present address: Allied Chemical. Syracuse Research Laboratory, P.O. Box 6, Solvay, NY 13209. 3 Present address: Department of Agronomy, United States Department of Agriculture, Science and Education Administration, Agricultural Research, University of Kentucky, Lexington, KY 40506. Samples Used in Test of Procedure. Corn (Zea mays L., Dekalb XL-45) seedlings were grown for 10 days under artificial light on half-strength Hoagland solution (4) which contained 7.5 mM ['4NJnitrate. Immediately after harvesting, the tissue was frozen with Dry Ice. The frozen tissue was then lyophilized and finally ground to a fine powder. Ten g ground shoot tissue were extracted with three successive 500-ml aliquots ofMCW4 (13:4:3 by volume, see reference 12). The extract was separated into two phases, chloroform and methanol:water. The chloroform phase was discarded. The methanol:water phase was surface-aerated overnight at room temperature to remove methanol. The remaining liquid, which contained [14N]nitrate, organic nitrogen, and some dissolved methanol, was used for the subsequent procedural tests. Nitrate in the liquid was determined using the method of Cataldo et al. (2). Stem exudate was collected from 5-day-old, dark-grown com seedlings (DeKalb XL-45) which had been cultured on a minusN nutrient solution (6) for 3 previous days. The resulting exudate was nitrate-free (verified by the method of Cataldo et al. (2), data not shown) and was used in the tests as collected. A salt solution of (NH4)2SO4 and KNO3 was also used in the various tests for comparison. Reduction of Nitrate during Digestion. The extent to which nitrate was reduced during Kjeldahl digestion was tested by adding selected quantities ofKlNO3 to each of the three kinds of samples previously described. The final '5N enrichment of nitrate was approximately 50 atom %. Appearance of '6N in reduced N was used to indicate that reduction of nitrate had occurred during digestion of the sample. The atom per cent excess 15N observed in the reduced N fraction was used to calculate the total quantity of nitrate (14N + 15N) that had been reduced. The Kjeldahl procedure of McKenzie and Wallace (8) was followed, using 2.5 ml 36 N H2SO4, 1.5 g K2SO4, and 50 mg HgO as HgSO4. Samples were digested in a heating block for 1 h at 200°C to remove H20. The temperature was then increased and the samples were digested for an hour after the block reached 380°C (total time approximately 3 h). Ammonium was determined directly on the digestate (3). Nitrate Volatilization. The proposed procedure depends on the removal of nitrate by volatilization as nitric acid from concentrated H2SO4. The completeness of nitrate volatilization was tested by heating selected quantities of KNO3 (25-250 ,umol) with 6 ml 3 N H2SO4 at 97°C for 48 h. The nitrate contained 49 atom % 15N so that a sensitive inverse isotope dilution procedure could be used to detect the trace amounts of nitrate remaining. This consisted of adding 5 ,tmol unenriched KNO3 (0.372 atom % 15N) to each 4 Abbreviations: MCW, methanol:chloroform:water; SRN, soluble-reduced N. 32 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1982 American Society of Plant Biologists. All rights reserved. MINIMIZING NITRATE REDUCTION Table I. Nitrate Reduction during Kjeldahl Digestion of Selected Samples Samples containing unenriched nitrate and reduced N were subjected to Kjeldahl digestion, without the presence of reagents that promote nitrate reduction (e.g. salicylic acid). ['5N]Nitrate was added to each sample and the atom per cent excess of the Kjeldahl-N was used to calculate the quantity of nitrate ("5N + '4N) reduced. Values reported are means + SE. SampleN n15NEnrichment in Sample N Recovered as Sample Nitrogen 15N Enrichment m Kjeldahl-N Test Sample Reduced Nitrate Nitrate Kjeldahl-N Reduced Nitrate N N 1lmol atom % excess % Tissue Extract' 5 ml 13.2c 20.7 43.7 19.06 ± 0.21 98.3 48.5 ± 1.2 20 ml 53.0c 83.0 44.5 23.94 + 0.14 79.4 59.1 ± 0.8 Stem Exudateb I ml 17.1 5.0 48.3 3.76 + 0.16 98.3 28.2 + 1.4 1 ml 17.1 20.0 48.3 5.96 + 0.04 98.8 11.9 ± 0.3 1 ml 17.1 50.0 48.3 7.01 + 0.11 99.4 14.4 ± 0.3 Salt Mixture' (NH4)2SO4/KNO3 50.4 100 48.5 0.24 ± 0.01 70.8 0.18 + 0.01 ' Ten observations. b Five observations. 'Value obtained with SRN procedure.