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TREE LEAF BIOMARKERS FOR ATMOSPHERIC NITROGEN DEPOSITION S. MARSH1 , A. J. MILLER2 , X.-H. ZHANG3 and J. PEARSON1∗ 1

Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK; 2 Crop Performance & Improvement Division, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK; 3 Shanghai Jiao Tong University, 2678 Qixin Road, Shanghai 201101, P.R. China (∗ author for correspondence, e-mail: [email protected])

Abstract. The aim of this paper was to investigate the effects of nitrogen (N) deposition on tree N cycling and identify potential biomarkers for N deposition. Between April and October 2002 extensive fieldwork was undertaken at Mardley Heath in Hertfordshire. This woodland, located adjacent to the A1(M) motorway, is exposed to high levels of atmospheric nitrogen oxides from the traffic. Measurements of δ 15 N, in vivo nitrate reductase (NR) activity, tissue, xylem and surface nitrate concentrations as well as N concentration and growth were made along a 700-m transect at 90◦ to the motorway. The δ 15 N data show that oxidised N from the road traffic is taken up by nearby trees and is incorporated into plant tissues. Our measurements of NR activities suggest elevated rates close to the motorway. However, xylem sap, leaf tissue and leaf surface nitrate concentrations showed no differences between the roadside location and the most distant sampling point from the motorway. Taken together the δ 15 N and nitrate reductase data suggest uptake and assimilation of N through the foliage. We conclude that for this lowland deciduous woodland, tissue, xylem and surface measurements of nitrate are unreliable biomarkers for N deposition whereas δ 15 N, growth measurements and integrated seasonal NR might be useful. The results also point to the benefit of roadside tree planting to screen pollution from motor vehicles. Keywords: Crataegus monogyna, nitrate reductase, nitrogen pollution, total nitrogen, Sambucus nigra

1. Introduction The global contribution of oxidised N from motor vehicles is in the region of 25 Tg N per year (Fowler, 2004). With many plant species being adapted for growth on soils with low N availability, this extra N may have significant effects on plant productivity and health. For instance, surveys of European forests have revealed increases in tree biomass accumulation (Kauppi et al., 1992). There is a need for biological markers of N deposition for the early detection of effects and changes to vegetation and habitats, particularly N-sensitive sites. In this context, biological markers (or biomarkers) can be defined as measurable changes that occur within the plant that can be directly related to a particular stress, in this case the deposition of N oxides. Cheap and readily available, plant biomarkers may provide an excellent means of tracing N deposition in the environment. The ideal would be to have an easily measurable, unambiguous response that is specific to oxidised N deposition (Jones and Coleman, 1989). Of course, in the natural environment plants are exposed to multiple simultaneous stresses that make finding such a specific biomarker very Water, Air, and Soil Pollution: Focus 4: 241–250, 2004. C 2004 Kluwer Academic Publishers. Printed in the Netherlands.

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difficult. In addition, intrinsic differences between species mean that the same biomarker may not always operate in the same way. Plant responses to oxidised N deposition are numerous and varied (for review see Welburn, 1990). Potential biomarkers could be changes in plant structure, function or metabolite pools. When N enters via the leaves the plant’s usual N uptake route from soil by the roots is circumvented. Physiological perturbations may result directly from damage caused by N uptake or be the result of adjustments that the plant makes to accommodate the additional N. Atmospheric oxides of N can be disposed of by assimilation into plant tissues following reduction by the enzyme NR (Nussbaum et al., 1993). Accompanying such an increase in assimilation may be changes in leaf concentrations of N-containing compounds along with adjustments in N transport between the root and shoot. Additional N may also promote growth. The aim of this work was to examine the effects of oxidised N on tree shoots and to identify potential biomarkers for oxidised N deposition.

2. Materials and Methods Trees were sampled at Mardley Heath in Hertfordshire (grid reference TL245185). This small woodland is adjacent to the A1(M) motorway. Sampling was primarily conducted at two sites at either end of a transect running in the prevailing wind direction (south–west) from the motorway to the control site 700-m downwind. The tree species sampled were Sambucus nigra L. and Crataegus monogyna Jacq. These species were selected because of their abundance at both sampling sites and to allow comparison between species that have different N uptake and assimilation strategies (Soares et al., 1995). S. nigra is very nitrophilous, utilizing mainly nitrate as an N source, and most of the assimilation occurs in the shoots. In contrast, C. monogyna is less able to use nitrate as an N source (Soares et al., 1995). Sampling took place approximately once a month during the growing season in 2002, beginning on 14th April. Leaf material and xylem sap was collected from branches approximately 1.5 m above ground and was kept cool in freezer bags for transfer to the laboratory. Xylem sap was collected into Eppendorf tubes using a hand-held vacuum pump (Rennenberg et al., 1998). This was also taken back to the laboratory where it was stored frozen before analysis. The NR activity of fresh leaves was assayed using the in vivo method of Stewart and Orebamjo (1979). Leaves were washed in 0.01 M HCl to remove surface contaminants. For examination of tissue metabolites, chopped unwashed leaf material was placed in methanol for extraction. The nitrate concentrations were determined enzymatically in vitro, using NR and assaying the resulting nitrite (Woodall and Forde, 1996). To assess N concentration, leaves were oven dried before being digested in a mixture of sulphuric acid and hydrogen peroxide (Allen et al., 1974). The ammonia produced by this digestion was measured using the method of McCullough (1967). Natural abundance of 15 N

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was determined by an automated C and N analyser coupled to a mass spectrometer (Barrie and Lemley, 1989). With the exception of the growth data, all samples were taken from three separate trees at each location. For the growth data, the average of 15 measurements of current year shoot extension was taken for a total of five trees. The data is presented as the average for the five trees at each location.

3. Results and Discussion Biological material can vary in δ 15 N because of variations in the initial signature of soil N sources (nitrate, ammonium, organic N) and subsequent fractionation processes due to uptake, assimilation and translocation (for background see Gebauer et al., 1994; Handley and Scrimgeour, 1997). Less is known about the δ 15 N signatures of N gases from anthropogenic sources. We were unable to measure the δ 15 N value of the NO2 source at the A1(M) site. However, Ammann et al. (1999) measured the δ 15 N signature of NO2 adjacent to a highway in the Swiss Middle land and found this to be +5.7‰. Figure 1 shows the effect the motorway traffic emission is having on the δ 15 N of C. monogyna. Close to the motorway the δ 15 N becomes less negative. Similar results were found for Quercus spp., Betula pendula, Carpinus betulus and Acer pseudoplatanus (data not shown). From this we can infer that N originating from the motorway vehicles is entering the leaf tissue and that the source signature is more positive than potential soil sources and subsequent fractionation processes. This is in agreement with the work of Pearson et al. (2000) who demonstrated that the δ 15 N of moss tissue close to roadsides

Figure 1. δ 15 N of Crataegus monogyna leaf material along the sampling transect. Points are an average of three replicates. SE bars are shown.

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Figure 2. Mean foliar in vivo NR activities in trees close to the motorway (solid line) and at the control site (broken line). (a) Sambucus nigra (b) C. monogyna. Data points are the average of three replicates. SE bars are shown. Sampling began on 14 April 2002 (day 1).

tended to be more positive than mosses sampled further away from traffic exposure. Oxidised N mainly enters plant leaves via the stomata, with a much smaller proportion entering through the cuticle (Zeevaart, 1976). Both NO and NO2 will dissolve in extracellular fluid to form nitrate, nitrite and protons (Lee and Schwartz, 1981). Nitrate and nitrite are able to pass through membranes into the mesophyll cells where they can be assimilated using the normal modes of N metabolism (Rogers et al., 1979). Figure 2 compares seasonal profiles of the leaf NR activity at the motorway and the control site for both species. Measurements of NR activity might be a good biomarker because, not only is it substrate inducible, but it may be the rate-limiting step in nitrate assimilation (Guerrero et al., 1981). From these


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data, the higher activities close to the motorway can be attributed to greater nitrate availability at this site. By integrating the area under the curves and subtracting the control from the motorway site, an estimate of the potential N input to the trees from the traffic can be obtained. This amounted to 2.6 and 1.4 kg N per hectare of leaf material per season for S. nigra and C. monogyna, respectively. Substrate availability is not the only factor that determines the activity of NR. Species differences and time of year also affect activity and consequently the data show a great deal of seasonal variation. S. nigra exhibits peak NR activity at the start of the growing season whereas activity in C. monogyna is highest just prior to senescence (Figure 2). Spring peaks can be related to high levels of soil nitrification and stored N remobilisation, whereas late seasonal peaks may allow the plant to convert inorganic N into organic form in preparation for next year’s growth (Clough, 1993). Species differences are also very apparent with S. nigra exhibiting much higher activities than C. monogyna in general. High leaf NR activities are characteristic of fast-growing pioneer species such as S. nigra (Pearson and Soares, 1995; Smirnoff and Stewart, 1985). Large seasonal and diurnal variations in activity mean that single readings of NR activity cannot be used as an unambiguous biological marker for oxidised N deposition (Norby, 1989). However, our data suggest that more extensive sampling, integrating leaf NR activity over the whole season, may have more potential as a biomarker for N deposition. Weekly measurements on a few carefully selected species would be feasible, given the ease of use of the leaf NR assay, and might also provide an assessment of direct N uptake by plants and potentially damaging eutrophication. The substrate for this enzyme, nitrate, was measured in the leaf tissue, the xylem supplying the leaves and on the leaf surfaces. The seasonal data for tissue nitrate are shown in Figure 3, and again there are marked seasonal variations with concentrations peaking in early spring when buds are beginning to break. As with the NR activity, nitrate concentrations are once again higher in S. nigra than in C. monogyna. This can be explained by the different N assimilation strategies of the two plants. As a fast growing pioneer, S. nigra transports nitrate to the shoot for assimilation. By this strategy, pioneers have more closely linked C and N acquisition in the leaf, when compared with climax species that carry out most of their N assimilation in their roots (Pearson et al., 2002). A preference for shoot N assimilation, high NR activity and a closer coupling between N and C metabolism in the leaf may give pioneer species such as S. nigra a greater buffering capacity against atmospheric inputs when compared with climax species like C. monogyna (Pearson and Soares, 1995). If oxidised N deposited on leaves enters through the stomata and subsequently enters the mesophyll cells as nitrate, we may predict concentrations of tissue nitrate to be highest close to the motorway. However, in Figure 3 there are no marked differences between tissue nitrate concentrations at the motorway and at the control site. Similarly we did not observe significant differences between the two sites with respect to xylem nitrate (Figure 4) and leaf surface

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−1 Figure 3. Mean tissue nitrate concentrations (mg NO− 3 /g fwt ). Data points are the average of three replicates. SE bars are shown. Sampling began on 14 April 2002 (day 1).

nitrate (data not shown). Amounts of nitrate on the leaf cuticle were very low at both sites. Figure 4 shows the seasonal profile of xylem nitrate in S. nigra. Trees are able to regulate N uptake in relation to shoot N demands (Rennenberg et al., 1998). Our data shows no inhibition of nitrate transport to the shoots despite the availability of extra N to tree leaves close to the motorway. However, it must be noted that because we do not have information regarding the transpiration rate, our measurements of xylem nitrate may not represent actual nitrate flux to the shoots. S. nigra is a highly nitrophilous species, and for this fast growing species, N from the motorway may be a welcome supplement to the plants’ total N supply. From Figures 3 and 4 it appears that the deposition of atmospheric N oxides appear to be having little or no significant effect on the amount of nitrate transported and stored in tissues. Despite this we have observed increased NR activity in trees near to the motorway (Figure 2). The fate of the motorway oxidised N was investigated by measuring the N concentration and growth. Figure 5 shows the N concentration in leaf material of S. nigra and C. monogyna measured from June 2002. Other studies have demonstrated a positive correlation between N concentration and atmospheric N in trees (Port and Thompson, 1980) and in mosses (Pitcairn et al., 1995). In S. nigra we observed a slight increase in N concentration of the leaves at the motorway site, but this is not statistically significant. No increase in N concentration was found in C. monogyna sampled


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−1 Figure 4. Mean nitrate concentrations (mg NO− 3 /ml ) in the xylem sap of S. nigra trees close to the motorway (solid line) and at the control site (broken line). Data points are the average of three replicates. SE bars are shown. Sampling began on 14 April 2002 (day 1).

Figure 5. Mean total leaf nitrogen concentrations (mg N/g dwt−1 ). Data points are the average of three replicates. SE bars are shown. Sampling began on 14 April 2002 (day 1).

from the motorway site. This result is not so surprising since according to our estimates of N inputs, C. monogyna is absorbing far less N from the atmosphere than is S. nigra. This suggests that leaf N concentration cannot be used as a reliable biomarker in all tree species. Since N is an essential plant nutrient that frequently limits growth, we might predict that an increase in N will result in enhanced growth. Kauppi et al. (1992)

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Figure 6. Histogram showing the average shoot extension over the growing season at the motorway and the control site. Data points are the average of five replicates. SE bars are shown.

showed that sulphur and nitrogen deposition were having fertilizing effects on many European forests. Figure 6 shows measurements of shoot extension during 2003 in both species. Both shoot growth and number of nodes (data not shown) are greater at the motorway site. The extra N available from motorway vehicles appears to be promoting growth in both species. However, the more nitrophilous species, S. nigra shows more growth than C. monogyna at the motorway site relative to the downwind site (Figure 6). An increase in plant size such as this will result in N dilution within the plant. This may explain why we did not observe significant differences with respect to leaf N concentrations.

4. Conclusions The δ 15 N data are consistent with the hypothesis that oxidised N from the motorway is taken up by nearby trees and is incorporated into plant tissues. This is in agreement with our measurements of leaf tissue NR activities that show elevated rates of nitrate assimilation close to the motorway. The two species investigated appear to be able to survive elevated N deposition without major physiological perturbations and may be useful for screening the effects of N oxides along roadsides. The results at this lowland deciduous woodland show that the N inputs are acting as a fertiliser, favouring the growth of more nitrophilous species, S. nigra. N deposition appears to be having little effect on the amounts of nitrate found on leaf surfaces and within leaf tissues. However, the δ 15 N and the NR activity data suggest that the uptake and assimilation of NO2 through the foliage is occurring. We can therefore conclude that measurements of tissue and surface nitrate are unreliable biomarkers for N


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deposition whereas δ 15 N, growth measurements and integrated seasonal NR might be more useful.

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