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The latter part of the 20th century saw rapid increases in the rates of reactive atmospheric nitrogen (N) deposition over much of Europe, and although deposition rates in the UK are now declining it remains at historically high levels (NEGTAP, 2001). Increases in N deposition have been implicated in declining floral diversity of species-rich seminatural grasslands, many of which have undergone considerable species change in response to experimental applications of simulated pollutant N (Van Dam et al., 1986; Willems et al., 1993; Bobbink et al., 1998; Carroll et al., 2003). These findings have heightened concern for the long-term sustainability of some of our most species-rich grasslands, especially as a high proportion of these have already been lost due to agricultural ‘improvement’ by nitrogen and phosphorus fertilizer additions (Rich & Woodruff, 1996).
The mechanisms by which reactive N deposition causes loss of some species but not others from grasslands, are incompletely known. There is evidence that in some cases, major floristic changes have occurred mainly due to a ‘fertilizer’ effect of N pollution, such as the expansion of Brachypodium pinnatum at the expense of forbs in Dutch calcareous grasslands. Combined with efficient uptake and retention of P by B. pinnatum, this grass showed enhanced growth and therefore increased shading and competitive exclusion of other species (Bobbink, 1991; Willems et al., 1993). Less clear, however, are the mechanisms of diversity loss in situations where N deposition has not led to increased productivity of the vegetation. For example, in calcareous grasslands on thin Rendzina soils, plants show little or no productivity gains in response to reactive N deposition since these ecosystems are primarily P limited, even before experiencing N pollution, and are therefore readily ‘nitrogen saturated’ (Jeffrey & Pigott, 1973; Willems et al., 1993; Carroll et al., 2003). Furthermore, there is now emerging evidence that in many N saturated ecosystems, high inputs of N have increased P limitation (Bobbink & Lamers, 2002).
In the case of calcareous grassland, simulated pollutant N has been found to decrease rather than increase productivity and to exacerbate P limitation, possibly through impaired cycling of P (Johnson et al., 1999). This may have important consequences for species composition, since it has been suggested that the balance between N and P supply may be critical for the maintenance of ecosystem diversity (Roem & Berendse, 2000). A fuller understanding of the impact of pollutant N on species-rich calcareous grasslands clearly requires the effects on the processes of P acquisition by the main functional groups of plants in these ecosystems to be established. Calcareous grasslands on Rendzina soils have exceptionally low concentrations of plant available P (Jeffrey & Pigott, 1973), and the main fund of the element is present as organic P compounds in the shallow organic-matter-rich surface horizons. The effect of pollutant N on the processes of organic P mineralization and the uptake of P by plants in calcareous grasslands is therefore a central issue for conservation of these increasingly rare, floristically rich communities.
Plants are dependent upon mineralized phosphate as their source of P (Duff et al., 1994) and this is acquired directly from soil solution by plant roots or through their mycorrhizal associates. In addition, inorganic P can be mineralized from organic P by root-surface, mycorrhizal, or soil and microbial phosphatases (Duff et al., 1994; Tarafdar & Marschner, 1994). Root-surface phosphatases may originate from root exudates, mycorrhizas or bacteria (Marschner, 1995). These enzymes include phosphomonoesterases, phosphodiesterases and phosphotriesterases (Florkin & Stotz, 1964). The expression of root-surface phosphomonoesterase (PME) activity is known to be closely regulated by, and a sensitive indicator of, the extent of P limitation to plant growth (Goldstein et al., 1988a, 1988b). In the presence of adequate supplies of orthophosphate, activity of the enzyme is repressed, whereas under P limitation it is stimulated. It has been inferred that root-surface PME plays an important role in P acquisition by plants (Duff et al., 1994; Marschner, 1995) and plant P uptake from specific phosphomonoesters such as inositol hexaphosphate is sometimes strongly correlated with activity of the enzyme (Kroehler & Linkins, 1988; Helal, 1990).
There is now increasing evidence that one of the major impacts of pollutant N on calcareous grasslands is through effects on the P nutrition of the plants. In a previous study of the effect of simulated pollutant N on root PME activity in a calcareous grassland, Johnson et al. (1999) found highly significant increases in the enzyme on roots of Plantago lanceolata seedlings only 7–13 d after transplantation into plots that had received 14 g N m−2 yr−1 for periods of between 18 months and 5 yr. The addition of orthophosphate fertilizer alone, and in combination with the N additions to plots treated for 18 months, reduced phosphatase activity of the seedlings and removed the stimulatory effect of N additions on the enzyme. The increases in PME activity showed strong positive correlations with soil extractable ammonium and shoot nitrogen concentration and a negative correlation with soil extractable phosphate (Johnson et al., 1999).
Crucial questions arising from this study are whether the responses to simulated N deposition seen in P. lanceolata are typical of other plants; if some plant functional types are more sensitive than others to P stress induced by N enrichment; and whether the effects increase over longer periods of N inputs.
Nitrogen treatments to the plots established by Johnson et al. (1999) have been maintained over a period of 8 yr and during this time there has been a gradual shift in plant community composition with an increase in grass and sedge cover at the expense of forbs. These three functional groups of plants differ in their adaptations to acquire P and this is likely to affect their different responses to N enrichment. The main forbs in calcareous grasslands are typically rather course rooted and heavily colonized by arbuscular mycorrhizal fungi, which play a major role in their P acquisition, whereas the grasses produce very fine roots with long root hairs and are less dependant upon mycorrhizas for P uptake (Read et al., 1976; Harley & Harley, 1987). The sedges typically lack mycorrhiza but produce specialist dauciform roots adapted for P uptake (Grime, 2001).
In this study we set out to test the hypothesis that representatives of these three plant functional types would show increases in root-surface PME activity in response to longer-term (7-yr) enhanced N deposition, and that activity of the enzyme is stimulated by higher soil and plant N concentrations, and decreased by addition of P to soil. We also hypothesized that since sedges – and to some extent grasses – have little or no dependence upon mycorrhizas for the uptake of P, and instead are reliant upon their root adaptations (dauciform roots/prolific root hairs), representatives of these plants will have higher PME activity, with greater responsiveness to pollutant N, than a typical forb.
The hypotheses were tested by transplanting bioassay seedlings of a forb (Leontodon hispidus), a grass (Koeleria macrantha) and a sedge (Carex flacca) into turf from a calcareous grassland that has received long-term N enrichment with simulated pollutant N deposition. The species were selected as they are common at the field site and are representative of the functional types’ contrasting root architecture and mycorrhizal dependence. The bioassay seedlings growth was then monitored over 14 and 28 d along with root-surface PME activity and shoot and soil nutrient status.
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The results confirm that root-surface PME activity, in representatives of three major higher-plant functional types of calcareous grassland, are highly sensitive and rapidly responsive to the effects of long-term enhanced N deposition. In all three species PME activity was significantly increased, more than doubling in each case, following only 14 d exposure to N treated soil. Similarly, with the exception of L. hispidus, the additions of P dramatically inhibited PME activity even at the first harvest.
The consistent and large effects on PME activity contrasted with the variable, and typically non-significant, effects of both the long-term N treatments and P additions on shoot biomass. The shoot N and P concentrations also showed very modest responses to the treatments compared to those of PME, even though in several species the enzyme activity correlated with these concentrations. The lack of major effects on shoot biomass, and the modest effects on shoot nutrient concentrations may have been in part due to the bioassay plants being slow-growing. This could be partially caused (and further confounded) by competition for, and uptake of, nutrients by the surrounding sward vegetation which could ‘dampen’ the treatment experienced by the bioassay seedling.
Our results extend the observations, made earlier in the treatment history of the same plots, of increased PME activity in roots of Plantago lanceolata after 13 d growth in the N enriched soil (Johnson et al., 1999). The present study importantly demonstrates that the root-surface PME activity of the grass, sedge and forb are all stimulated in a dose-dependant manner by the N treatments, although activity of the enzyme differs by an order of magnitude between the species. Nonetheless, the PME activities lay well within the reported range of values for plant roots (e.g. 0.08–420 nmol g−1 s−1; Lee, 1988; Helal, 1990; Johnson et al., 1999). Since phosphatase is located on the root surface and the enzyme activities are expressed per unit root weight, differences in root surface area to volume ratios of the different species – a characteristic of the functional types to which they belong – most simply explain the different activity rates. For example, C. flacca and particularly K. macrantha have very fine roots and long root hair lengths, characteristic of grasses and sedges, and consequently will have much higher area to volume ratios than the typically course-rooted forb, L. hispidus. The low rates of PME activity in L. hispidus therefore do not reduce the chance of observing treatment effects or correlations with soil and shoot nutrient data, since these low rates are predominantly a result of expressing rates per root f. wt and not of low absorbance values for (and poor resolution of) PME activities during the enzyme assay.
We hypothesized that the three functional types of plants would differ in the magnitude of their responses to P stress induced by simulated pollutant N deposition. This is not clear in the direct responses to the N treatments where all three species showed consistently large increases in PME activity. However, the results clearly show that the three species differ considerably in the magnitude of response to direct changes in alleviation of P limitation (i.e. response to the + P treatment) with responses being greatest in the sedge and grass, with no response apparent in the forb. The greater PME activity and sensitivity to P supply in the grass and sedge than the forb interestingly correspond with the observation from the N treated plots of a loss of cover of forbs and relative increase in cover of sedges and grasses (P. Horswill, G. Phoenix and J. Hodgson, unpublished data). Further work is clearly required to establish whether the floristic changes are due to a direct causal relationship between the root-surface PME activity of species and their tolerance to P limitation in N polluted grassland ecosystems.
PME responses are also likely to be related to the mycorrhizal status of the plants. We did not determine the mycorrhizal status of the bioassay seedlings, but the large differences in mycorrhizal colonization of established plants of the same species growing in the untreated soil will almost certainly have occurred in the seedlings by the 28 d harvest. The established plants of L. hispidus had high rates of mycorrhizal colonization and their broad tap root with relatively little branching indicates that this species will be heavily dependant upon mycorrhizas for P uptake, and its roots poorly adapted for direct uptake of P from the soil (Read et al., 1976; Hetrick, 1991). The PME activity in this species was least sensitive to changes in shoot and soil nutrient status as judged by the limited number and weak strength of correlations between these factors, together with its low enzyme activity per unit biomass. Most notable was the lack of significant reduction in PME activity in this species in the P treated soil. By contrast, PME activities of K. macrantha, and especially the often non-mycorrhizal C. flacca, were very strongly affected by P treatments and more sensitive to changes in shoot and soil nutrient status. K. macrantha, while mycorrhizal, has a finely branching root system with prolific root hairs and has a much lower rate of mycorrhizal colonization than L. hispidus and is almost certainly less reliant upon mycorrhiza for P uptake (Hetrick, 1991). Therefore, at least in the species used in our study, it would seem that the more mycorrhizal dependant the plant species is, and the less adapted the roots are for direct P uptake, the less sensitive root-surface PME activity is to direct changes in P limitation.
Although we confirm that root-surface PME activity is highly sensitive to soil N and P treatments we do not know what fraction of root-surface PME activity originates from root exudates or root associated mycorrhizas and bacteria. Furthermore, the functional significance of the enzyme is still unclear. It is not known whether the higher activity in the N enriched soil has any benefit for P nutrition of the plants, although the negative correlations between shoot P concentration and enzyme activity in C. flacca and K. macrantha indicates tight regulation of activity in these species. However, even if PME does play an important role in plant P nutrition, it is clear that the + 14 N treatment results in high N : P ratios in some seedlings (particularly in K. macrantha, day 28) so it would seem that stimulation of this enzyme alone is not enough to redress the nutrient imbalance of plants in severely N polluted systems.
The inhibitory effects of P additions on PME activity are well established and known to arise both as a consequence of repression of enzyme production together with competitive inhibition of the active site by orthophosphate (Kroehler & Linkins, 1988; Duff et al., 1994; Hunter & McManus, 1999). However, the positive relationships between PME and soil or shoot N status seen in C. flacca and L. hispidus, in the present study, and in P. lanceolata in the earlier study by Johnson et al. (1999) suggest that root-surface PME activity may not be exclusively controlled by shoot and soil P status, though it should be noted that these correlations cannot be used to prove a controlling role for N. In our study, the correlations observed were relatively weaker (lower r2 values) than those observed by Johnson et al. (1999); the weaker correlations resulting from a large variation in enzyme activity compared to the more modest changes in shoot and soil nutrient status that the treatments caused. Goldstein et al. (1988a, 1988b) suggested that stimulation of phosphatase production before P limitation of growth is detectable is a ‘phosphate starvation inducible’ stress response. This pre-empts phosphate starvation by upregulation of phosphatase gene expression to increase the efficiency of uptake of P from the environment and the recapture of P leakage from tissues. In our study, stimulation of PME activity by the N treatments often occurred in the absence of convincing evidence of P limitation to growth, since addition of P to the soil significantly increased the shoot weight of seedlings only in the case of K. macrantha at day 28. Therefore, most of the stimulatory effects of N on PME activity may be phosphate starvation inducible type responses, and our results suggest that the expression of genes in the proposed phosphate starvation rescue system could be sensitive to shoot and soil N as well as P.
Johnson et al. (1999) observed strong correlations between soil extractable ammonium and root-surface PME activity in Plantago lanceolata but no correlation with soil nitrate, and the same results were obtained from Agrostis capillaris seedlings introduced into a parallel series of N-treated plots on an acidic grassland. It was suggested that ammonium was more important than nitrate for stimulating root-phosphatase activity but the ammonium concentrations were much higher and varied more widely than nitrate showing 180-fold and 2-fold variation, respectively. In the present study, variation in extractable ammonium and nitrate concentrations were much more comparable (day 14: ammonium 0–25 µg g−1, nitrate 0–22 µg g−1; day 28: ammonium 0–64 µg g−1, nitrate 0–22 µg g−1), and whilst some positive relationships were found between PME activity and soil nitrate, we found no significant relationships with soil ammonium.
Attempts to elucidate the mechanisms behind ecosystem change resulting from pollutant N have justifiably concentrated on direct effects of N treatments (e.g. shoot N concentrations, nitrate reductase activity, frost hardiness, soil N mineralization/nitrification; Caporn et al., 1994; Carroll et al., 1999; Pearce & Van der Wal, 2002). However, if ecosystem responses to N pollution are to be fully understood, particularly in systems which may be or are becoming increasingly P limited, the indirect effects of pollutant N on plant P nutrition certainly warrants further examination.