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FOLIAR NITRATE REDUCTASE: A MARKER FOR ASSIMILATION OF ATMOSPHERIC NITROGEN OXIDES Richard J. Norby Environmental Sciences Division Oak Ridge National Laboratory Oak Ridge, Tennessee 37831-6034 ABSTRACT Atmospheric deposition of nitrates to forest canopies has been implicated as predisposing trees to environmental stresses. A first step in the evaluation of this hypothesis is the determination of whether atmospheric nitrates are absorbed and metabolized by tree foliage. The enzyme nitrate reductase (NR) is an appropriate marker for nitrate metabolism because it is the rate-limiting step in the assimilation of nitrate into organic compounds and it is substrate-inducible. In laboratory studies, NR was not induced in red spruce foliage exposed to NO3- in acid mist, but NR activity increased dramatically in spruce seedlings exposed to NO2 or HNO3 vapor, suggesting that gaseous nitrogen oxides can be assimilated by spruce foliage. Nitrate reductase activity also can be measured in the field and may be a useful marker for trees that are impacted by nitrogen pollution, but extensive characterization of diurnal and seasonal variation in foliar NR activity is an important prerequisite. THE NITROGEN HYPOTHESIS OF FOREST DECLINE One of the hypotheses that has been put forth to explain forest decline in eastern North America and western Europe is the so-called "nitrogen hypothesis." This hypothesis states in part that elevated levels of nitrogen deposition in a form available for direct foliar uptake by tree canopies (e.g., HNO3 vapor, NO, N02) disrupts normal nutrient cycles and physiological processes, resulting in increased susceptibility to other stresses (Lindberg et al. 1987). Foliar absorption of gaseous nitrogen oxides (NOX), in contrast to root uptake from the soil solution, circumvents the plant's regulatory control of N uptake, increasing the possibility of physiological imbalances. The possible physiological imbalances include disruption of the metabolic and growth processes required for winter hardening (Nihlgird 1985, Friedland et al. 1984, Waring 1987) and changes in carbon allocation patterns (McLaughlin 1985). An unfavorable balance between transpiring leaves and water-absorbing roots can increase drought susceptibility 1 Research sponsored by the USDA, National Acid Deposition Assessment Program Interagency Agreement 40-1647-45 with the U.S. Department of Energy under contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. Publication No. 31 13, Environmental Sciences Division. 245
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246 (Norby et al. 1986~. Imbalances in other nutrients can also result (Mohren et al. 1986, Waring 1987~. A BIOLOGICAL MARKER FOR EVALUATION OF NITROGEN HYPOTHESIS The nitrogen hypothesis is difficult to test experimentally because of the number of steps between the predisposing stress (NOx) and the response to the inciting stress (drought or winter conditions). A co-occurrence of NOx deposition and forest decline clearly is insufficient evidence for a causal relationship. An efficient approach might be to investigate the critical links in the logical sequence of necessary events linking atmospheric deposition to changes in whole-plant physiology (Lindberg et al. 1987~. One such link is the metabolism of foliar absorbed NOx: if the nitrogen compounds are not reduced and assimilated into organic compounds, an alteration of carbon-nitrogen relations and the attendant effects on stress susceptibility are unlikely. The reduction of nitrate to nitrite by the enzyme nitrate reductase (NR) is the rate-limiting step in the assimilation of nitrate into organic compounds (Guerrero et al. 1981~. The activity of this enzyme may, then, be an appropriate marker for determining whether foliar-absorbed nitrogen oxides are assimilated by tress. This paper will explore the possible use of nitrate reductase as a marker in manipulative laboratory studies as well as in field surveys, with a focus on the impact of nitrogen deposition to high elevation red spruce (Picea reubens) forests. CHARACTERISTICS OF NITRATE REDUCTASE Nitrate reductase is potentially a valuable biological marker because it is substrate- inducible and responsive to environmental parameters. The presence of the substrate (nitrate) causes the de novo synthesis of the enzyme, an uncommon phenomenon in higher plants (Zielke and Filner 1971~. Because the turnover rate of the enzyme is rapid (4-h half-life under noninducing conditions, Zielke and Filner 1971), the amount of active enzyme in tissue is low unless its synthesis is induced. The presence of NR in tissue, however, is not diagnostic for nitrate uptake because nitrate is not an obligatory inducer (Guerrero et al. 1981~. There is a certain low level of NR always present ("constitutive NR") that is functional in nitrate reduction but not influenced by nitrate concentration (Beevers and Hageman 1983, Rajasekhar and Oelmlbller 1987~. Furthermore, other N compounds can induce NR, including ammonium (Rajasekhar and Oelmlkller 1987), nitrite, urea, and certain amino acids (Haynes and Goh 1978~. The activity of NR is influenced by a number of other environmental factors, including light, temperature, drought, and time of day, and physiological factors, such as hormones, carbohydrates, leaf water potential, and tissue age (Beevers and Hageman 1983~. Rapid variations in NR activity in response to these factors are not due to long-term changes in the amount of enzYme. but are the result of inactivating proteins (Guerrero et al. 1981~. With these precaut~ons and exceptions in mind, it can, nevertheless, be stated that the level of NR is generally high in organisms grown on nitrate and low in organisms grown on ammonium (Guerrero et al. 1981~. Although nitrate is not an absolute requirement for the induction of NR, the presence of active NR is an absolute requirement for the reduction, and, hence assimilation, of nitrate. There are many conflicting generalizations in the literature regarding the N nutrition of forest trees, and it is difficult to predict the level of NR that might be present in the foliage of a given tree. There appears to be no universal correlation between environmental factors and root or leaf assimilation of nitrate (Smirnoff and Stewart 1985~. Plants vary in their dependence on ammonium vs. nitrate, in large part because of conditions in the rooting media, but most plants, including trees, can induce NR and assimilate nitrate if nitrate is present (Smirnoff et al. 1984~. Under low levels of nitrate uptake by trees, most of the nitrate