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247 may be reduced in the roots (Andrews 1986), nevertheless, NR activity commonly is found in the leaves of many woody plants (Smirnoff et al. 1984, Al Gharbi and Hipkin 1984~. Foliar NR activity in gymnosperms generally is low, but it can be induced by nitrate feeding (Smirnoff et al. 1984~. Yandow and Klein (1986), however, reported that NR was not measurable in needles of red spruce seedlings growing with or without nitrate. If red spruce needles have no or very low NR activity, then NR would need to be induced before foliar-absorbed nitrate could be assimilated. The research question becomes whether NR is indeed induced in red spruce needles when they are exposed to nitrogen oxides. EXPERIMENTAL USE OF NITRATE REDUCTASE AS A MARKER One characteristic of NR that is important to its potential use as a biological marker is that it is fairly easy to assay. The so-called in viva assay, which actually uses chopped tissue and not whole intact plants, obviates difficult enzyme extraction and purification steps. The protocol, which is derived from the approach described by Jaworski (1971), should be optimized for each species or tissue type (Al Gharbi and Hipkin 1984~. A spectrophotometer is the only instrument required, so the assay could be done in the field. Alternatively, branch samples can be brought back to the laboratory if certain precautions are taken. It is important to note that the NR assay does not determine the rate at which nitrate is being reduced in intact tissue. Total extractable NR activity greatly exceeds the actual in situ rate; the enzyme is present in excess and is operating below its Vmax. The in vivo assay predicts the capability for reduction (Huffaker and Rains 1978, Andrews 1986~. We have conducted a series of laboratory experiments to determine whether NR is induced in red spruce needles after exposure of the plants to NO2, HNO3 vapor, and NOB- in mist (Norby et al., in prep.~. NR activity in needles of seedlings exposed to 75 ppb NO2 increased rapidly within 1 day after the fumigation began, stayed at a level about twice that of control seedlings during the fumigation, and dropped quickly back to control levels 1 to 2 days after the NO2 was withdrawn. No nitrate or nitrite accumulated in the needles. Very similar responses were obtained with exposure to KNOB vapor (about 75 ppb), except recovery was slower. The slower recovery possibly was due to continued movement (and hence continued induction of NR) of surface-adsorbed HNO~ into the leaf interior (Marshall and Cadle 1987~. There was an indication of nitrate (but not nitrite) accumulation in the needles exposed to HNO3 vapor. Unlike the gaseous nitrogen oxides, nitrate applied in pH 3.5 mist did not increase NR activity over that in seedlings exposed to pH 5.0 mist. The results of the experiment with NO2 fumigation are similar to the results of Wingsle et al. (1987), who exposed Scots pine seedlings with 85 ppb NO2 and detected a large increase in foliar NR activity after 1 day, with maximum activity after 2-4 days of exposure. Similar experiments with HNO3 vapor have not been reported. These results show that red spruce has the capability to assimilate gaseous nitrogen oxides. Foliar NR was a useful marker for this purpose. EVALUATION OF NITRATE REDUCTASE AS A MARKER IN THE FIELD The use of NR as a biological marker has not been tested in the field. We excavated several red spruce seedlings from an intensively-studied field site in the Great Smoky Mountains National Park (McLaughlin et al. 1988), and exposed them to NO2 along with the greenhouse-grown plants described above. Although the level of NR activity in

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248 both treated and control plants was lower than in the greenhouse-grown seedlings, the pattern of response to NO2 was quite similar (Norby et al., in prep.~. Nitrate reductase also was assayed on cut shoots of mature spruce trees from two sites in the Smoky Mountains. The samples collected from the higher elevation site had 50% higher NR activity on September 24 and 30h higher on October 15 compared to samples from the lower elevation site (Y. Weerasuriya and R. J. Norby, unpublished data). The high elevation samples also exhibited a greater capacity for NR induction, as shown by putting the cut stems in KNO3 solution. The NR activity of noninduced needles ranged from 20 to 110 nmol NO2-/(h~g DW), which is similar to that reported for noninduced needles of other conifers (Smirnoff et al. 1984) and is typical for constitutive NR (Al Gharbi and Hipkin 1984~. The level of NR activity of samples collected on November 11 was much lower than that of the previous collections, and there was no difference between sites. There is, not yet, a firm basis for speculation about why the spruce trees differed between sites in their levels of NR or in NR inducibility. The differences could be attributable to different carbon economies, phonology, soil characteristics, nitrogen deposition, or random variation. One of these sites is adjacent to a site described by Johnson et al. (1988) as having very high levels of nitrate in the soil solution, although foliar N concentrations of the red spruce trees are only about 1 Ho. Much more extensive monitoring of the trees during the entire growing season will be necessary before an explanation might emerge. The results do show, however, that mature red spruce trees have NR in the needles, albeit at very low levels, that foliar NR is inducible, and that foliar NR activity in seedlings is responsive to NO2 and HNO3 vapor. Whether pollutant NOX gases cause a detectable increase in foliar NR at realistic concentrations and under field conditions remains a key research question. The literature on NR suggests that it is unlikely that a simple measurement of NR activity in the foliage of a tree, or even a more extensive monitoring of changes in foliar NR of a tree during the season or during a suspected pollution episode, will provide an unambiguous marker that the tree is impacted by nitrogen oxides. There are too many other environmental and physiological factors in addition to nitrate concentration that also induce NR or regulate its activity. Furthermore, some level of NR can be expected always to be present (constitutive NR), which may be sufficient to metabolize most foliar-absorbed NOX without altering the marker. SUMMARY 1. Nitrate reductase, which is the key enzyme involved in assimilation of NOB,- by plants, is relatively easy to assay in the foliage of trees, can be detected at very low levels in the foliage of mature, forest-grown trees, and is known to respond to the amount of nitrate in tissue. It may provide an appropriate marker for evaluation of an important aspect of the "nitrogen hypothesis." 2. Nitrate reductase is an appropriate biological marker in controlled, manipulative experiments to test whether foliage has the capacity to metabolize foliar-deposited nitrogen oxides. 3. In such experiments it has been demonstrated that red spruce seedlings do have the capability for foliar assimilation of NO2 and KNOB vapor, but wet-deposited nitrate did not affect the marker. Additional experiments on the effect of foliar-absorbed N on

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249 whole-plant physiology hypothesis." are, therefore, appropriate for evaluating the "nitrogen 4. Extensive monitoring of NR activity during a growing season and in relation to realistic levels of NOX pollutants will be necessary to evaluate the utility of NR as a biological marker in the field, but it probably will not prove to be an unambiguous marker for an impact of nitrogen pollutants. REFERENCES Al Gharbi, A., and C.R. Hipkin. 1984. Studies on nitrate reductase on British angiosperms. New Phytol. 97: 629-639. Andrews, M. 1986. The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ. 9: 511 - 519. Beevers, L., and R.H. Hageman. 1983. Uptake and reduction of nitrate: Bacteria and Higher Plants. Pp. 351 -375 in A. Lluchli and R.L. Bieleski (eds.), Encyclopedia of Plant Physiology, New Series Vol. 15A. Springer-Verlag, Berlin. Friedland, A.J., R.A. Gregory, L. Karenlampi, and A.H. Johnson. 1984. Winter damage to foliage as a factor in red spruce decline. Can. J. For. Res. 14: 963-965. Guerrero, M.G., J M. Vega, and M. Losada. 1981. The assimilatory nitrate-reducing system and its regulation. Ann. Rev. Plant Physiol. 32:169-204. Haynes, R.J., and K M. Goh. 1978. Ammonium and nitrate nutrition of plants. Biol. Rev. 53: 465-510. Huffaker, R.C., and D.W. Rains. 1978. Factors influencing nitrate acquisition by plants; assimilation and fate of reduced nitrogen. Pp. 1-43 in D.R. Nielson and J.G. MacDonald (eds.), Nitrogen in the Environment, Vol. 2. Academic Press, New York. Jaworski, E.G. 1971. Nitrate reductase assay in intact plant tissue. Biochem. Biophys. Res. Commun. 43: 1274- 1279. Johnson, D.W., A.J. Friedland, H. Van Miegroet, R.B. Harrison, E. Miller, S.E. Lindberg, D.W. Cole, D.A. Schaefer, and D.E. Todd. 1988. Nutrient status of some contrasting high-elevation forests in the eastern and western United States. In G.D. Hertell, chairman, Effects of Atmospheric Pollutants on the Spruce-Fir Forests of the Eastern United States and Federal Republic of Germany. Proceedings of a symposium; 1987 October 19-23; Burlington VT. Gen. Tech. Rep. NE-??, U.S. Dept. Agric., For. Serv., Northeastern For. Expt. Sta., Broomall, PA. in press. Lindberg, S.E., G.M. Lovett, and K-J. Meiwes. 1987. Deposition and forest canopy interactions of airborne nitrate. Pp.117- 130 in T.C. Hutchinson and K.M. Meema (eds.), Effects of Atmospheric Pollutants on Forests, Wetlands and Agricultural Ecosystems. NATO ASI Series, Vol. G16. Springer-Verlag, Berlin. McLaughlin, S.B. 1985. Effects of air pollutants on forests. J. Air Pollut. Control Assoc. 35: 512-534.

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250 McLaughlin, S.B., C.P. Andersen, P.J. Hanson, R.J. Norby, N.T. Edwards, and R.R. Tardiff. 1988. Interactive effects of natural and anthropogenic factors on growth and physiology of southern red spruce. In G.D. Hertell, chairman, Effects of Atmospheric Pollutants on the Spruce-Fir Forests of the Eastern United States and Federal Republic of Germany. Proceedings of a symposium; 1987 October 19-23; Burlington VT. Gen. Tech. Rep. NE-??, U.S. Dept. Agric., For. Serv., Northeastern For. Expt. Sta., Broomall, PA. in press. Marshall, J.D., and S.H. Cadle. 1987. Cuticular deposition of nitric acid vapor and its assimilation by eastern white pine. General Motors Research Report EV-294. Warren, MI. Mohren, G.M.J., J. Van den Burg, and F.W. Burger. 1986. Phosphorus deficiency induced by nitrogen input in Douglas fir in the Netherlands. Plant Soil 95: 191-200. Nihlgard, B. 1985. The ammonium hypothesis - an additional explanation to the forest dieback in Europe. Ambio 14: 2-~. Norby, R.~., G.E. Taylor, Jr., S.B. McLaughlin, and C.A. Gunderson. 1986. Drought sensitivity of red spruce seedlings affected by precipitation chemistry. Pp. 34-41 in C.G. Tauer and T.C. Hennessey (eds.), Proceedings, Ninth North American Forest Biology Workshop, Stillwater, Oklahoma. Rajasekhar, V.K., and R. Oelmlbller. 1987. Regulation of induction of nitrate reductase and nitrite reductase in higher plants. Physiol. Plant. 71:517-521. Smirnoff, N., P. Todd, and G.R. Stewart. 1984. The occurrence of nitrate reduction in the leaves of woody plants. Ann. Bot. 54: 363-374. Smirnoff, N., and G.R. Stewart. 1985. Nitrate assimilation and translocation by higher plants: comparative physiology and ecological consequences. Physiol. Plant. 64: 133- 140. Waring, R.H. 1987. Nitrate pollution: a particular danger to boreal and subalpine coniferous forests. Pp. 93- 105 in T. Fujimori and M. Kimura (eds.), Human Impacts and Management of Mountain Forests. Forestry and Forest Products Research Institute, Ibaraki, Japan. Wingsle, G., T. Nasholm, T. Lundmark, and A. Ericsson. 1987. Induction of nitrate reductase in needles of scots pine by NOX and NO3-. Physiol. Plant. 70: 399-403. Yandow, T.S., and R.M. Klein. 1986. Nitrate reductase of primary roots of red spruce seedlings. Plant Physiol. 64: 723-725. Zielke, H.R., and P. Filner. 1971. Synthesis and turnover of nitrate reductase induced by nitrate in cultured tobacco cells. J. Biol. Chem. 246: 1772-1779.