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OCR for page 245
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
OCR for page 246
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
Representative terms from entire chapter:
nitrogen oxides
