Nitrate and Nitrite Reduction by Wolffia arrhiza

Abstract

Nitrate reductase was not found to be present in or associated with partially purified, intact chloroplasts aqueously isolated from Wolifia arrhiza. Such chloroplasts are capable of using nitrite but not nitrate as an electron acceptor during light-stimulated electron transport in the absence of additional cytoplasmic components. When nitrite acts as an electron acceptor under these conditions, on the average 1.5 moles of oxygen are evolved per mole of nitrite reduced by the chloro-plasts, indicating a probable reduction of nitrite to ammonia. Chloroplasts ruptured by osmotic shock fail to reduce nitrite in the absence of additional components. The subcellular distribution of nitrate reductase and nitrite reductase in leaves is still a matter of controversy and has not been clearly resolved. Although it was demonstrated first by Evans and Nason (6) and more recently by Del Campo et al. (5) and Losada et al. (10) that nitrate can act as a terminal electron acceptor in a light-driven reaction in the presence of grana, FMN, NADPH, and nitrate reductase, it does not necessarily follow that such a reaction occurs in vivo. In fact, Ri-tenour et al. (12) concluded from studies of chloroplasts isolated aqueously and nonaqueously from Zea mays L. and Setari fabrii Herrm. that nitrite reductase was located inside the chloroplasts but that nitrate reductase was not. In contrast to this report, Coupe et al. (4) using nonaqueous density gradients found that nitrate reductase was associated with barley chloro-plasts, although no marker enzymes were used. The possibility was advanced by Beevers and Hageman (2) that nitrate re-ductase is associated with the chloroplast envelope, and they suggested that further studies are needed to resolve the conflicting results. The present work was undertaken to determine the ability of isolated intact chloroplasts of Wolffia arrhiza to reduce nitrate and nitrite. MATERIALS AND METHODS Nitrite Determination. Nitrite was determined by a modification of the colorimetric method of Wooley et al. (15) in which 2 ml of 0.5% sulfanilamide, 0.75 N HCI, 0.04% N-(1-naphthyl)ethylenediamine dihydrochloride were added to an aliquot of the plant homogenate. After color development for 5 min, the reaction mixture was centrifuged, and the absorbance of the supernatant was determined at 540 nm. Nitrate Reductase. It was found that in order to determine nitrate reductase in W. arrhiza by the method of Beevers et al. (3), a modification of the grinding and assay media was necessary. Two grams of fresh plant material were ground in 1.0 ml of Jensen and Bassham's (9) solution A with a VirTis model 45 homogenizer. The homogenate was strained through nylon mesh and centrifuged at 17,000g for 5 min. An aliquot of 0.5 ml was added to 0.5 ml of 0.02 M KNOS in solution C, pH 7.6 (9). The reaction was started by adding 0.5 ml of 1.36 mM NADH in solution C, pH 7.6. A blank was prepared by substituting 0.5 ml of solution C for the NADH. After 30 min of incubation at 24 C the reaction was stopped by adding the color reagent for nitrite, and the absorbance at 540 nm was determined. Isolation of Chloroplasts. Chloroplasts were isolated by Jen-sen and Bassham's (9) method as modified by Robinson and Stocking (13), and the percentage of intact chloroplasts was estimated by measuring the rate of light-dependent NADP-stim