Atmospheric Environment Vol. 32, No. 3, pp. 533—538, 1998 ( 1998 Elsevier Science Ltd All rights reserved. Printed in Great Britain 1352—2310/98 $19.00#0.00
PHYSIOLOGICAL RESPONSES OF PLANT LEAVES TO ATMOSPHERIC AMMONIA AND AMMONIUM J. PEARSON* and A. SOARES Department of Biology (Darwin Building), University College London, Gower Street, London WC1E 6BT, U.K. (First received 6 November 1995 and in final form 2 December 1996. Published February 1998) Abstract— Misting of leaves of several plant species with 3 mM aqueous NH` at pH 5, or fumigation with 4 3000 kg m~3 gaseous NH for 1 h, elicits similar biochemical and physiological changes in the species 3 tested. The enzyme glutamine synthetase (GS) was shown to increase its activity in all species, while that of nitrate reductase (NR) was inhibited, at least in those species which possessed the ability to induce foliar NR. At the same time there were marked changes in organic anion concentrations, with malate and citrate in particular being reduced in concentration, following either NH` or NH application to leaves. The 4 3 changes in organic anions are also discussed in the light of pH regulation by the cell. A stimulation of photosynthesis was also evident when leaves were treated with either NH or NH`. It is argued that, 3 4 because of the differences in solution chemistry of the two ammonia forms, the aqueous form applied at pH 5 and the gaseous form being an alkali in solution, these changes can only have occurred through the ability of the leaves to readily assimilate both forms of the ammonia. The biochemical changes might have potential as markers for the onset of physiological perturbation by atmospheric ammonia pollution, particularly changes in organic acid concentration; their use in an index of pollution stress is briefly discussed. ( 1998 Elsevier Science Ltd. All rights reserved. Key word index: Ammonia, photosynthesis, pH regulation, organic acids, glutamine synthetase, nitrate reductase, bioindicator.
An understanding of the biochemical and physiological perturbations that an organism may undergo when subject to an atmospheric pollutant, such as ammonia, is of fundamental importance. This understanding can be used for early diagnosis of potential damage (Saxe, 1991); specific biochemical or metabolic markers might be monitored to test for the onset of perturbations (Wild and Schmitt, 1995). A greater knowledge of cause and effect can be used for dose—response studies, which in turn can be allied to an approach based on critical loads and levels abatement. This approach acknowledges that it may be impossible to completely remove a pollutant, but possible to limit it to some threshold level below which no damage occurs. For example, species or possibly varieties may be more or less susceptible to a pollutant (Soares et al., 1995). Armed with this information planting policies in agriculture or arboriculture may be altered, or ecosystems with particularly vulnerable species may be more closely monitored. In short, a greater awareness of the effect of a particular pollu-
* Author to whom correspondence should be addressed. Fax: 0171 380 7096; E-mail: [email protected]
tant can lead to more effectual controls or treatment of pollution damage. In practice, the study of cause and effect of pollution can be quite complex. This is mainly because of the complicated biology of organisms, with a myriad of biochemical pathways, many of which may be interlinked. It is, therefore, often a difficult task to trace biochemical disturbance directly to a particular pollutant. It must also be remembered that atmospheric pollution often occurs against a complicated background of atmospheric chemistry and nearly always involves a mixture of pollutants which can confound interpretation of biochemical changes. Nevertheless, some advances in our understanding of the effects of ammonia pollution on the biochemistry of plants have recently occurred. For example, uptake of ammonia by roots will reduce cation uptake (van Dijk and Roelofs, 1988); sugars and organic acids are known to decline (Soares and Pearson, 1996); photosynthesis is thought to be stimulated, or at least unaffected by atmospheric ammonia (Wellburn, 1994); and certain amino acids are known to increase in concentration, among them arginine in some conifers (van Dijk and Roelofs, 1988). This list is by no means exhaustive and several reviews cover these topics, and others, in greater detail (Fangmeier et al., 1994; Pearson and Stewart, 1993).
J. PEARSON and A. SOARES
However, little work has been carried out which considers the effect of both gaseous ammonia or aqueous ammonium on plant biochemistry; that is, do ammonia or ammonium elicit the same biochemical responses? In this paper we use short-term applications of either gaseous ammonia or aqueous ammonium to shoots of a range of plant species expected to differ in their intrinsic nitrogen metabolism (Pearson and Soares, 1995). In particular, we are interested in the effects of foliar uptake, and so treatments were excluded from impacting on the soil in which the plants were grown. The physiological responses of the plants to these treatments are discussed in relation to their value as potential markers for atmospheric ammonia pollution.
Figure 1 shows the response of the enzymes glutamine synthetase (GS) and nitrate reductase (NR) in the leaves of four species that were misted with 3 mM NH Cl solution. GS activity was increased 4 with the NH` treatment in all four species. The in4 creases were statistically more significant (P(0.001) in the two species with a relatively low control GS activity, G. hederacea and P. padus. The response of NR activity in the same four species, however, was different. In P. padus and Q. robur very low NR activities ((0.3 kmol h~1 g~1 FW) were found in controls and the misting treatment did not significantly affect these low rates. However, the two remaining species had control NR activities that
2. MATERIALS AND METHODS
All plant species, with the exception of Glechoma hederacea (ground ivy), which was treated in situ, were grown in a greenhouse at 20°C. Three tree seedlings: 2 yr old Populus deltoides (poplar); 3 yr old Prunus padus (bird cherry) and 3 yr old Quercus robur (pedunculate oak), were planted in to 30 cm pots with a loam-based compost, peat and sand (mixed in the ratio 1 : 1 : 1), and the plants allowed to establish for two months prior to treatment. Phaseolus vulgaris cv tendergreen (French bean) was grown from seed sown in to a loam-based compost in 20 cm pots and grown for 4 weeks prior to treatment. Pots were watered regularly with distilled water. Plants were treated with NH either by misting with NH` x 4 or fumigation with NH . For misting with NH` plants were 3 4 placed inside a perspex cabinet fitted with a spinning disc nozzle mister. A solution of 3 mM NH Cl at pH 5 was 4 applied as a fine mist ((6 km droplet size) over a period of 20 min. The plants were left to dry (approximately 2—4 h) and sampling occurred 24 h after the treatment. Fumigation of plants with an estimated average of 3 mg m~3 NH was also 3 carried out inside a sealed perspex cabinet, which was fitted with a fan to aid mixing of the internal air. Gaseous NH was 3 generated from a solution of NH Cl which had strong alkali 4 added to start the fumigation, and plants left in the cabinet for 1 h before being removed to the greenhouse. After this period tests on the remaining solution showed that all the NH` had been converted to the gaseous form. During both 4 misting and fumigation treatments, the plant pot and stems were sealed with plastic bags to prevent the treatment affecting the soil. In a separate experiment 0.25 m2 field plots of Glechoma hederacea were misted with 500 ml of 3 mM NH Cl using a hand-held mister, and plants again sampled 4 24 h following treatment. Three replicates were sampled from separate control and treatment plants (unless otherwise stated) and statistical comparisons carried out by means of a t-test. Enzyme activities and metabolite concentrations were measured as described in Soares et al. (1995). For nitrate reductase (NR), 0.1 g of whole leaf tissue was assayed using the in vivo method. Photosynthesis was measured as CO 2 uptake using a portable infra-red gas analyser (IRGA) (model LDC4, ADC, Hoddesdon, U.K.). Additionally, a leaf disc oxygen electrode was used to measure O evolution, as 2 described by Walker (1993). For measurement of the effects of gaseous NH on photosynthesis, the NH , which was 3 3 taken from a cylinder of known concentration, was introduced into the electrode chamber using a syringe in order to give a final NH concentration of 4 mg m~3 inside the chamber. 3 Oxygen evolution was then monitored for 2 min following the introduction of NH to the chamber. 3
Fig. 1. The effect of a 3 mM NH Cl misting treatment on 4 the activities of inorganic nitrogen assimilation enzymes in the leaves of four plant species: GS—glutamine synthetase, NR—nitrate reductase (in vivo). Enzyme activities were measured 24 h following the treatment. Controls (open bars) were misted with double-distilled water; NH Cl misting 4 (solid bars). Values are the means of three replicates with standard deviation (vertical lines). Significant differences between treatments: NS—not significant; *P(0.05; **P(0.01; ***P(0.001.
Physiological responses of plant leaves
were at least an order of magnitude greater ('3 kmol h~1 g~1 FW) and both showed marked inhibition of NR with NH` treatment, e.g. P. deltoides 4 showed a reduction of activity from about 8 kmol h~1 g~1 FW in controls to around 5 kmol h~1 g~1 FW with the NH` treatment. The 4 two major organic anions in all four species were malate and citrate, one, or both, of which showed significant decreases in concentration when the leaves were treated with NH` (Fig. 2). 4 The changes in enzyme activity described above were also found when P. vulgaris was either misted with NH`, or fumigated with NH (Fig. 3). 4 3 In both instances GS activity increased and NR activity declined, although the increase in GS with NH fumigation was not statistically significant. 3 Similarly, both NH` or NH treatments resulted in 4 3 a decline in malate concentration, but again the
Fig. 2. Changes in organic anion content of leaves of four species misted with 3 mM NH Cl. Details as described under 4 Fig. 1. Controls — open bars, NH Cl misted — solid bars. 4 Values are the means of three replicates with standard deviation (vertical lines). Significant differences between treatments: NS—not significant; *P(0.05; **P(0.01; ***(0.001.
Fig. 3. The effect of misting leaves with NH Cl or fumiga4 tion with NH on glutamine synthetase (GS) activity, nitr3 ate reductase activity (NR) (in vivo), or malate content of leaves of Phaseolus vulgaris. Samples were measured 24 h after the treatment. Misting was with 3 mM NH Cl. Fumi4 gation was carried out in a closed cabinet with 3000 kg m~3 NH for 1 h. Controls — open bars, treatments — solid bars. 3 Values are the means of three replicates with standard deviation (vertical lines). Significant differences between treatments: NS—not significant; *P(0.05; **P(0.01; ***P(0.001.
J. PEARSON and A. SOARES Table 1. The effect of treatment of leaves with NH and NH` on photosyn3 4 thesis. Phaseolus vulgaris was misted with a solution of 3 mM NH Cl and 4 photosynthesis followed with either an IRGA (CO uptake) or an oxygen 2 electrode (O evolution) 24 h following treatment. Photosynthesis in leaves of 2 Populus deltoides was measured as O evolution over a 2 min period of 2 following introduction of 4 g m~3 NH directly in to the oxygen electrode 3 chamber. Values are the means of 6 replicates $ SD Species and treatment
Flux of CO or O (kmol m~2 s~1) 2 2 Control
CO 2 O 2
! 8.27$0.16 #17.95$1.22
Phaseolus vulgaris 3 mM NH Cl 4 Populus deltoides 4 g m~3 NH 3
statistics showed this was not significant for the NH 3 fumigation. Changes in photosynthesis were monitored by two means, IRGA and O electrode. In P. vulgaris treated 2 with 3 mM NH Cl both techniques showed that the 4 treatment enhanced photosynthesis (Table 1). The discrepancy in gas flux between the two techniques is due mainly to the need to use 5% CO in the leaf disc 2 O electrode chamber, a concentration that sup2 presses photorespiration (Walker, 1993). The O elec2 trode was also useful for measuring photosynthesis in very short-term experiments. P. deltoides was treated with a very high, but short-term (2 min) exposure to 4 g m~3 NH , the effect of which was to significantly 3 increase photosynthesis under these conditions (Table 1).
The enzyme responsible for ammonia assimilation in higher plants is GS. In the leaves of four species misted with NH` a significant increase in GS activity 4 was found (Fig. 1). The regulation of GS is complex and not fully understood, although some evidence for substrate NH` induction of GS does exist (for dis4 cussion see Forde and Woodall, 1994). The data presented here seem to support this, but even in these short-term treatments the effect of added N on amino acid and protein/enzyme production cannot be ignored. Treatment of P. vulgaris leaves with either NH` or NH also caused total GS activity to increase 4 3 (Fig. 3). Although a relatively high dose of NH 3 (3000 kg m~3) was used to fumigate P. vulgaris leaves, and the treatment was only short-term, the results agree with more realistic long-term fumigations of Scots pine ( Pe´rez-Soba et al., 1994). Scots pine fumigated with a lower NH (60 kg m~3) or a high NH 3 3 (240 kg m~3) concentration induced GS activity by some 25—70% over a period of 6—12 weeks of fumigation, when compared to filtered air controls
(Pe´rez-Soba et al., 1994). However, changes in enzyme activity alone should be treated with some caution, the activities in vitro may not reflect those in vivo. Additionally, because there is often an already high rate of GS activity in leaves, due to photorespiratory turnover of ammonia (Wallsgrove et al., 1983), any increase in activity resulting from uptake of atmospheric ammonia may be masked by a potentially high rate of photorespiration (Pearson and Stewart, 1993). There are additional problems in interpreting the response of GS to NH applicax tion in that light is also known to regulate GS activity (Woodall et al., 1996) and in greenhouse or field situations this may also be the cause of some variation in GS activity. With this in mind we can note in Fig. 1 that G. hederacea and P. padus both had relatively low control GS activities, which increased more markedly with NH` treatment than 4 in the remaining two species with higher control GS activities. P. vulgaris treated at the same age but in two separate experiments also showed that control activities could vary, unfortunately the light intensity at the time of the experiments was not monitored, but is likely to account for some of these differences (Fig. 3). GS also exists as several isoforms in most species, the exact function and tissue and cell compartmentalisation we are only just beginning to understand (Woodall et al., 1996). Thus, the data also raise the question of which of the two main isoforms of GS (chloroplastic or cytosolic) is affected? Preliminary work in our laboratory on P. vulgaris showed both isoforms induced to some extent with gaseous NH 3 treatment (not shown). The degree of variation in GS activity, even in the same species, has been remarked by Pe´rez-Soba et al. (1994); such variability makes it an unfavourable biochemical marker for NH pollution. x The inhibition of NR by NH` is a well documented 4 response, when plant roots are treated with NH` 4 (Pearson and Stewart, 1993), and foliar uptake of
Physiological responses of plant leaves
NH` elicits a similar inhibition (Figs 1 and 3). This 4 reduction of shoot NR activity with NH` treatment 4 was also apparent in three species of moss (Soares and Pearson, 1997). However, it is noticeable in Fig. 1 that two species were not affected by NH` treatment, 4 P. padus and Q. robur. Undoubtedly, this is due to their already low control NR activity (Pearson and Soares, 1995). Since these species can be classed as plants that prefer root assimilation of inorganic or organic nitrogen, fertilising these species with nitrate does not greatly induce foliar NR activity (Pearson and Stewart, 1993; Pearson and Soares, 1995; Soares et al., 1995). In P. vulgaris fumigated with NH , the 3 leaf NR activity was also inhibited. The inhibition of NR is, therefore, common to both NH` and NH 4 3 treatments. There is more of an open question about the exact mechanism of inhibition of NR by ammonia assimilation. Amino acids as end products have been suggested as inhibiting the enzyme (for a discussion see Pearson and Stewart, 1993). An alternative explanation may lie in the change in organic acids, particularly malate. Ben-Zioni et al. (1971) suggested that a potassium malate shunt may serve as a mechanism for nitrate transport between root and shoot. Treatment of leaves with ammonia reduces the malate content (Fig. 2), and may in turn prevent or reduce the efficiency of the malate shunt, thus reducing nitrate uptake by the roots. Even those species which have a very low capacity to reduce nitrate in their leaves show decreases in malate and citrate concentrations when misted with NH` (Fig. 2). A decline in the organic acid content of 4 leaves with ammonia treatment might be interpreted as the organic acids supplying carbon skeletons for incorporation of ammonia to organic form. However, it is well documented that organic acids tend to increase in concentration when nitrate is reduced and incorporated into organic molecules, a process that inevitably occurs via reduction to, and subsequent assimilation of, ammonia (Pearson and Stewart, 1993). Alternatively, the changes in organic acid concentration may also occur as part of a mechanism to maintain cell pH homeostasis. Reduction of nitrate to ammonia by NR and nitrite reductase generates OH~, whereas assimilation of ammonia generates H`; even with assimilation of ammonia generated by nitrate reduction, net OH~ is produced (Raven, 1988). Similar responses have also been shown to occur in three species of moss misted with either NO~ or NH` 3 4 (Soares and Pearson, 1997). A further area that has generated some debate in the literature is the response of photosynthesis to atmospheric ammonia. Some workers suggest that photosynthesis is not affected by NH (Berger et al., 3 1986; Wellburn, 1994), others that it enhances photosynthesis (van der Eerden, 1992). The results presented in Table 1 suggest that both NH and NH` 3 4 may stimulate photosynthesis in the species tested. In P. vulgaris measurement of intact leaves using an
IRGA and in detached leaves using the O elec2 trode, lend support for a slight but significant stimulation of photosynthesis. This is also accompanied by a slight increase in the stomatal conductance (IRGA measurement, results not shown). P. deltoides when treated with NH also showed 3 a stimulation in O evolution. Admittedly, a very 2 high concentration of NH was used in this experi3 ment; it was found that 0.5 g m~3 was the minimum concentration to affect the leaf photosynthesis. However, we have already remarked that even for CO the 2 concentrations in the leaf chamber have to be high and the requirement for high concentrations of NH may be due to similar constraints on uptake by 3 the leaf in the chamber, probably because stomata are closed and uptake is via the cuticle (Walker, 1987). The degree of stimulation of photosynthesis was found to be species-dependent as well as concentration dependent. For example, longer term treatments with seven misting episodes of 1 mM NH` over a 28 4 day period led to a gradual inhibition of photosynthesis in P. vulgaris (Soares and Pearson, data not shown). The exact mechanism that may promote CO 2 uptake and assimilation is not clear (Pearson and Stewart, 1993). Considering the combined evidence from changes in GS and NR enzyme activities, and photosynthesis, along with those of the organic acids measured, one factor stands out, which is that NH or NH` both 3 4 induce the same biochemical and physiological changes in the plants sampled. In considering these consistent physiological changes it is helpful to consider the consequences of assimilation of ammonia for cell pH homeostasis. Uptake of both NH` and NH , 4 3 with subsequent assimilation, would lead to net generation of acidity (Raven, 1988). The differences in solution chemistry, the NH gas being an alkalising 3 agent, and the NH` being applied at pH 5, would 4 very likely preclude the changes being due to uptake and storage of ammonium alone. Indeed, measurements of free ammonia in total leaf homogenates of some of these species showed no evidence for the build up of a storage pool of ammonia in treated plants (data not shown). There is in fact little evidence for ammonia accumulation in plant tissues, and the already high GS activity, which can increase with NH treatment, may prevent just this occurrence. As x far as we know, this is the first time that such a similarity of changes of both enzyme activities and metabolite concentrations have been shown for both NH and NH`. 3 4 A consistent of response to conditions of the controlled environment (greenhouse temperature in this instance) may not always translate to conditions in the field. The possible problems in using GS as a marker enzyme for ammonia pollution were noted above (Pearson and Stewart, 1993). Diurnal and seasonal variation is often also confounded by choosing leaves of the same physiological development and age. However, a combination of fairly easily measured
J. PEARSON and A. SOARES
physiological parameters could be used to develop an index of pollution stress. For example, the organic anions malate and citrate respond in the way outlined in this paper across a wide range of species. As metabolites they also respond to both nitrogen uptake as well as cell acidity, these are of major interest in the study of atmospheric pollution in general (SO , acidity, etc.) and to ammonia pollu2 tion. Soares et al. (1995) have shown that the capacity of the leaf to buffer against acidity can be used as a means of determining plant susceptibility to acidic pollution. An index of pollution stress, therefore might be formulated by combining a measure of buffering capacity, with changes in metabolites, such as organic cations. Changes in amino acids could also incorporated, or replace part of the index, thus increasing the potential sensitivity of such an index by including metabolites that increase or decrease in concentration. An index of this form or combination of biochemical changes would then integrate the plant or species response to pollution and remove some of the problems involved with environmental variation. Acknowledgements — This work is funded by the U.K. Department of the Environment (Contract no. EPG1/3/14). We thank two anonymous referees and Dr Mark Sutton for useful comments.
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