INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

After N₂ and N₂O, NH₃ is the most abundant nitrogen species in the atmosphere (Raven et al., 1992) with NH₄⁺ being the dominant cation in precipitation in Europe and North America for the last four decades (Flechard, 1998). There is increasing evidence to suggest that where the atmosphere becomes artificially enriched with NH₃ there may be detrimental effects on sensitive (low nitrogen) ecosystems due to eutrophication. The best documented instances of eutrophication due to nitrogen deposition have been the replacement of ericaceous species, such as Calluna vulgaris and Erica tetralix, by grass species such as Molinia caerulea and Deschampsia flexuosa (e.g. Heil and Diemont, 1983; Roelofs, 1986; Leith et al., 1999). Furthermore, although NH₃ rapidly protonates to NH₄⁺ in solution, metabolism of NH₄⁺ by plants and microbes can lead to the acidification of poorly buffered ecosystems (van Breemen et al., 1982, 1983). Raven (1988) calculated that the uptake and assimilation of NH₃ by the aerial parts of plants leads to the generation of 0.22 H⁺ N¹, whereas uptake and assimilation of NH₄⁺ leads to the generation of 1.22 H⁺ N¹. Nitrification of NH₄⁺ by soil microbiota leads to the generation of 2 H⁺ N¹ (van Breemen et al., 1983), whereas there is a net generation of 1 H⁺ N¹ if deposition is as NH₃.

It was demonstrated as early as 1850 that plants can take up and utilize gaseous NH₃ (Ville, 1850; cited in Sutton et al., 1993). It has since become accepted that a stomatal NH₃ compensation point exists that determines, at a given atmospheric NH₃ concentration, whether NH₃ will be taken up by, or released from stomata (e.g. Farquhar et al., 1980; Lemon and Van Houtte, 1980). The NH₃ compensation point is thought to be determined by the pH and the NH₄⁺ concentration of gas exchange sites in the foliar apoplast (e.g. Farquhar et al., 1980; Husted and Schjoerring, 1995). As a consequence, there has recently been interest in measuring the pH and NH₄⁺ concentration of the foliar apoplast as a simpler method of determining the NH₃ compensation point than carrying out accurate measurements of gas exchange with foliage, which are considerably more time consuming. Apoplast pH has been measured by a variety of methods including the use of pH sensitive microelectrodes (Bowling and Edwards, 1984; Okazaki et al., 1995; Hanstein and Felle, 1999), equilibration with solutions (e.g. Raven and Farquhar, 1989; Pearson et al., 1998), and pH-sensitive fluorescent dyes (Pfanz and Deitz, 1987; Mühling et al., 1995). The use of pH-sensitive dyes, although not allowing the calculation of NH₃ compensation points, has provided data that suggest spatial heterogeneity of pH within the foliar apoplast.

Hanstein and Felle (1999) attempted to measure apoplast NH₄⁺ concentration in intact leaves using a microelectrode inserted through a stoma, but encountered problems of sensitivity and interference from K⁺. Extraction of the foliar apoplast by pressure vessel (Jachetta et al., 1986; Hartung et al., 1988) or by infiltration of leaves and extraction by centrifugation (e.g. Husted and Schjoerring, 1995; Hanstein et al., 1999) allows pH and NH₄⁺ concentration of extracted liquids to be measured. Only the infiltration and centrifugation extraction method has been used for the prediction of NH₃ compensation points to date.

It is important for estimation of the magnitude and direction of NH₃ fluxes that the NH₃ compensation point be determined accurately. This investigation made a comparison between the determination of NH₃ compensation points for a widespread plant of semi-natural habitats (Luzula sylvatica [Huds.] Gaud.) by calculation from the pH and NH₄⁺ concentration of apoplast extracts and by direct measurement of gas exchange. For the first time an evaluation of possible causes of discrepancies between compensation points determined by the two methods (e.g. Mattsson et al., 1997; Mattsson and Schjoerring, 1999) was made. Evaluation included the important issue of apoplast buffering, which has been assumed to be zero in previous determinations of NH₃ compensation points.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

Cytoplasmic Contamination

Comparison of the malate dehydrogenase activity of total leaf homogenates and apoplast extracts showed a mean cytoplasmic contamination of apoplast extracts of 0.14% with a maximum value of 0.86% when the homogenate of lowest activity was compared with the apoplast extract of highest activity.

Influence of Indigo Carmine on the pH and NH₄⁺ Analysis of Extracts

No significant difference (P  0.05) was found when the pH of apoplast extracts collected after infiltration with deionized water was compared by t test (assuming unequal variance, Microsoft Excel) with those collected after infiltration with 50 µM indigo carmine.

The presence of indigo carmine in standard NH₄⁺ solutions had no systematic effect on the analysis of the NH₄⁺ concentration (data not shown).

Osmotica

The five methods for estimating the osmolality of the foliar symplast gave widely varying estimates (Table I). It was considered that freezing caused the greatest cell disruption, although there was no statistical difference between boiling and freezing determinations (t test, P  0.05).

Estimates of the intercellular air space volume (Table II) were not significantly different when compared by single factor analysis of variance (P  0.05, Microsoft Excel). The estimate derived via infiltration with silicone fluid was the lowest. This may have been influenced by the difficulty of achieving complete infiltration of L. sylvatica leaves with the silicone fluid.

The NH₄⁺ concentration of apoplast extracts was not significantly different (t test, P  0.05) between those infiltrated with 50 g L¹ mannitol and those infiltrated with 91 g L¹ mannitol, although the non-significant difference between the means (Table II) was in the direction that would be expected if either the 50 g L¹ solution caused a flux of water from apoplast to symplast, or the 91 g L¹ solution caused a flux of water from the symplast to the apoplast.

As mentioned earlier, the uncertainty in the methods for symplastic osmolality determination and the lack of statistically significant differences in other tests made it preferable to use low osmolality solutions for infiltration.

Comparison of NH₃ Compensation Points Determined by Gas Exchange and Bioassay

Values for the NH₃ compensation point as determined by gas exchange measurements were among the lowest yet reported. Values of the NH₃ compensation point calculated from the pH and NH₄⁺ concentration of apoplast extracts were the lowest yet reported (Table III). Other reported controlled gas exchange compensation point measurements (25°C) range from 0.05 µg m³ (1 µg NH₃ m³ is approximately 1.5 nmol NH₃ mol¹ air at 1 atm and 25°C) for Arrhenatherum elatius (Hanstein et al., 1999), which is exceptionally low as compared with other gas exchange determinations, to >26 µg m³ for wheat (Morgan and Parton, 1989). Reported compensation points calculated from apoplast extracts range from 0.04 µg m³ for low-nitrogen maize (Loubet et al., 1999) to 36.2 µg m³ for high-nitrogen barley (Mattsson et al., 1998). In general, non-crop plants have been found to have lower NH₃ compensation points than crop plants (Morgan and Parton, 1989; Husted and Schjoerring, 1996; Husted et al., 1997; e.g. Hanstein et al., 1999; Mattsson and Schjoerring, 1999), although compensation point values (25°C) such as 0.3 for µg m³ low-nitrogen oilseed rape (Husted and Schjoerring, 1996; Schjoerring et al., 1998a), 5.9 µg m³ for Calluna vulgaris (Schjoerring et al., 1998b), and 0.03 µg m³ for maize (Loubet et al., 1999) are notable exceptions. Higher NH₃ compensation points in crop plants may reflect their adaptation to situations where nitrogen availability is high.

View this table: [in this window] [in a new window]   Table III.   Comparison of values of the NH3 compensation point and  as determined by gas-exchange measurements and apoplast extraction Where appropriate, errors are SEs.

A marked discrepancy was found between values of the NH₃ compensation point and  determined by gas exchange measurements and those determined from apoplast extracts (Table III; Fig. 1). NH₃ compensation points determined via gas exchange were between 2- and 30-fold higher than those calculated from apoplast extracts, with a mean difference of 5-fold. Such a discrepancy between compensation point values determined by the two methods can also be seen in the data of Mattsson et al. (1997) and Mattsson and Schjoerring (1999), with discrepancies ranging from 1.5- to 7-fold. In a similar manner, such a difference can be inferred from the data of Hermann et al. (1999), which shows sustained emission fluxes of NH₃, measured by micrometeorological methods, during periods when the calculated NH₃ compensation point was approximately 8-fold lower than the atmospheric NH₃ concentration. Sutton et al. (1999) drew attention to apoplast extract-based (Loubet et al., 1999) and micrometeorological determinations (Milford et al., 1999) of the NH₃ compensation point for a fertilized grassland in Southern Scotland that again show lower NH₃ compensation points calculated from bioassays than would be predicted from gaseous flux measurements. Husted and Schjoerring (1995, 1996) reported good agreement between compensation points determined by the two methods, whereas Hanstein et al. (1999) found compensation points calculated from apoplast bioassays to be 3- to 8-fold larger than those determined from gas exchange measurements. However, Hanstein et al. (1999) suggested that the discrepancy was caused by coelution of amines with NH₄⁺ in ion chromatograph measurements leading to an overestimation of apoplast NH₄⁺ concentration.

View larger version (28K): [in this window] [in a new window]   Figure 1.   Comparison of values of  (NH4+/H+) determined by gas exchange and by apoplast extraction. AC, Ammonium chloride; AN, ammonium nitrate.

Given that a discrepancy has been found between the two methods of determination in all investigations other than those of Husted and Schjoerring (1995, 1996) on oilseed rape, it appears likely that a flaw exists in the methodology or the interpretation of results. Two basic differences exist in the procedure used by Husted and Schjoerring (1995, 1996) for the calculation of NH₃ compensation points and that used here, (a) the correction for dilution of H⁺ during the extraction procedure, and (b) the correction of the NH₄⁺ dissociation constant (K_d) for the ionic strength of the apoplast. However, in both cases, utilization of the methods of Husted and Schjoerring (1995, 1996) leads to lower estimates of the NH₃ compensation point. Furthermore, the change in pH during the dilution of apoplast extracts from L. sylvatica suggested that the apoplast was sufficiently buffered to eliminate the need for correction of H⁺ concentration for dilution up to a factor of at least 2.5-fold (Fig. 2). The data of Nielsen and Schjoerring (1998) suggests that the pH of the apoplast of oilseed rape is similarly regulated and/or buffered.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Change in measured H+ concentration of an example apoplast extract with dilution (solid line) compared with the calculated H+ concentration if the extract were unbuffered (broken line).

Correction of K_d for the ionic strength of the apoplast gives estimates of the NH₃ compensation point and  approximately 0.9 of those presented here assuming an apoplast ionic strength of 20 mM calculated on the basis of measured ion concentrations (Table IV) and a predicted negative charge of 4.9 mM. Given that the unquantified negative charge was "added" as a monovalent species, the ionic strength of the apoplast may in reality be greater than 20 mM, and the calculated NH₃ compensation points lower than 0.9 of the values presented here.

Infiltration of leaves with solutions of differing pH also suggested that the apoplast was subject to almost complete homeostasis with respect to H⁺ concentration within the range of perturbations carried out here (Fig. 3). The change of the H⁺ concentration of solutions after infiltration was almost 1:1 correlated with the departure of the infiltrated solution from the apoplast H⁺ determined by infiltration with unadjusted indigo carmine solution (see gradient of measured data regression line, Fig. 3). Approximate homeostasis of the apoplast with respect to pH is likely to be due to apoplast buffering and transplasmalemma fluxes. Nielsen and Schjoerring (1998), in similar infiltrations of oilseed rape leaves, found the pH of the apoplast to be regulated. They considered the regulation only in terms of transplasmalemma fluxes of H⁺ rather than buffering, although the data presented here (Fig. 2) clearly show that buffering occurs in the apoplast of L. sylvatica. This is consistent with the reports of apoplast buffering made by Böttger et al. (1980), Pfanz and Dietz (1987), Ikoma and Okamoto (1988), Peters and Felle (1991), and Hanstein and Felle (1999).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Comparison of the departure of the H+ concentration of the infiltrated solution from that of the apoplast, with the difference of H+ concentration between the infiltrated solution and the extract ([H+]infiltration solution  [H+]extract) (solid line). The H+ concentration of the apoplast was assumed to be that of extractions performed with unadjusted indigo carmine solution. Values are means of 10 values ± SE. The broken line shows the theoretical values based on simple mixing of the apoplast with the infiltration solution.

Buffering would also be expected from the presence in the foliar apoplast of organic acids undergoing dissociation or protonation at physiological values of apoplast pH e.g. citrate, and malate (Table IV; Speer and Kaiser, 1991; Lohaus et al., 1995; Mühling and Sattelmacher, 1995; Gabriel and Kesselmeier, 1999).

Homeostasis of the Apoplast with Respect to NH₄⁺ Concentration

Infiltration of leaves with solutions of differing NH₄⁺ concentration, although suggesting that NH₄⁺ is transported from the foliar apoplast to the symplast, did not show complete homeostasis of the apoplast with respect to NH₄⁺ concentration in L. sylvatica within the approximate 4 min between infiltration and centrifugation (Fig. 4). However, Nielsen and Schjoerring (1998) reported that the NH₄⁺ concentration of the apoplast of oilseed rape was upwardly regulated to a value of 0.8 mM within 1.5 min after infiltration with an NH₄⁺-free solution. Such a rapid upward regulation was not taking place in leaves of L. sylvatica. Figure 4 shows that infiltration with a solution approximately equal to the NH₄⁺ concentration of the apoplast extract collected after infiltration with NH₄⁺-free indigo carmine solution caused an increase in the NH₄⁺ concentration of the resulting extract. It therefore appears that, despite the evidence for uptake of NH₄⁺ from the apoplast, a correction for the dilution of NH₄⁺ in the apoplast of L. sylvatica during the extraction procedure is necessary when calculating NH₃ compensation points.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Variation in the NH4+ concentration of extracts with the NH4+ concentration of the solution infiltrated into leaves. The broken line shows the theoretical NH4+ concentration of extracts if simple mixing was the only process involved. For this the concentration of the extract after infiltration with deionized water and correction for dilution was used as the apoplast NH4+ concentration. Values are means of 10 values ± SE Two regression lines are shown to allow for possible curvature in the plot. The linear regression is significant at P < 0.001. The curvilinear regression is a second order polynomial.

Errors in the Determination of NH₃ Compensation Points by Gas-Exchange Measurements

Gas-exchange measurements, being the more empirical method of compensation point determination, should theoretically give the most reliable estimates. No obvious sources of error existed in these measurements other than possible NH₃ exchange between the plant growing medium and the atmosphere. In control experiments with artificial plants measurable exchange of NH₃ did not appear to be taking place with the growing medium of experimental plants.

Cytoplasmic Contamination of Extracts

Cytoplasmic contamination of extracts is not a likely explanation for the observed discrepancy in the determinations of the NH₃ compensation points since such contamination would be likely to increase the pH and the NH₄⁺ concentration of apoplast extracts. Thus, calculated NH₃ compensation points would probably be overestimated.

Use of Osmotica in Infiltration Solutions

Assuming that infiltration of leaves with low osmolality solutions caused a flux of water from the apoplast to the symplast, it would be likely that indigo carmine concentrations would be higher than in the absence of such fluxes. Correction for dilution would therefore underestimate the proportion of the extract that was apoplastic in origin, and increase the factor by which NH₄⁺ concentrations were multiplied during correction for dilution. Again this would lead to an overestimation of the apoplast NH₄⁺ concentration and the NH₃ compensation point.

Loss of Indigo Carmine within the Leaf

If indigo carmine were adsorbed by surfaces within the leaf, there would be a tendency to overestimate the proportion of extracts that were of apoplastic origin. This could lead to an underestimate of the factor by which extract NH₄⁺ concentrations were multiplied to correct for dilution during extraction, and thus underestimation of the NH₃ compensation point. The use of indigo carmine in infiltrations to determine apoplast liquid volumes has precedent in the literature (Cosgrove and Cleland, 1983; Husted and Schjoerring, 1995, 1996; Husted et al., 1997; Mattsson et al., 1997; Mattsson et al., 1998; Nielsen and Schjoerring, 1998; Hanstein et al., 1999; Mattsson and Schjoerring, 1999). Furthermore, results have been validated by Husted and Schjoerring (1995) who compared results from indigo carmine infiltrations with [¹⁴C]sorbitol infiltrations and found good agreement between the two approaches. They did, however, find 2% of the indigo carmine in a 6-µM solution thereof adsorbed to isolated cell walls of oilseed rape. Such an adsorption would be insufficient to account for the discrepancy between NH₃ compensation points determined by gas exchange and those calculated from apoplast extractions. Cosgrove and Cleland (1983) also found good agreement between apoplast liquid volume determined by indigo carmine and [¹⁴C]mannitol in stems of pea and soybean, but suspected that some indigo carmine might be lost during infiltrations of cucumber. It should be noted that Cosgrove and Cleland (1983) reported averages of only two determinations with no indication of variation and that there is, as they point out, no guarantee that the [¹⁴C]mannitol determination was not responsible for the observed discrepancy.

It is possible that there may have been significant loss of indigo carmine during the infiltration and extraction process, although assuming apoplast pH was correct, the magnitude of the indigo carmine loss would have needed to be great to account for such large discrepancies in compensation points determined by the two methods used here. For instance, in a hypothetical extraction a 50-µM solution of indigo carmine was infiltrated into leaves and resulted in an extract indigo carmine concentration of 15 µM. If the concentration of indigo carmine in the extract without any loss should have been 25 µM, the correct calculated compensation point would only be 1.4 times higher than that calculated from the extract where dye loss had occurred. Therefore, although such loss of indigo carmine during the extraction process would lead to an underestimation of calculated NH₃ compensation points, it does not seem possible that it could have made more than a contribution to observed discrepancies.

Error in the Determination of Apoplast pH

Small errors in the determination of apoplast pH could potentially lead to much larger errors in calculated NH₃ compensation points due to the logarithmic relationship between pH and H⁺ concentration. However, it is clear that a large margin of error in the measurement of apoplast pH (e.g. 0.3-1.5 pH units) would be necessary to account for the magnitude of the discrepancies between the two methods of compensation point determination.

Rust Infection of Experimental Plants

Rust infection of experimental plants may have had an influence on the magnitude of the NH₃ compensation point. However, documented effects of rust infection suggest that the NH₃ compensation points of infected plants would be higher than those of uninfected plants. Tetlow and Farrar (1993) reported an alkalization of the apoplast of rust-infected barley plants as compared with controls, and Fukuda (1996) noted a similar extracellular alkalization from cultured cells in response to a fungal elicitor. Fungal infection has been linked to an increase in phenylalanine ammonia-lyase activity (Habereder et al., 1989; Hahlbrock and Scheel, 1989; Walter, 1989), which is one of the principal NH₃-generating enzymes in plants (Raven et al., 1992). Higher NH₄⁺ generation rates are consistent with reports of increased foliar NH₄⁺ concentrations (Farkas and Király, 1961) and increased volatilization of NH₃ (Jenkyn and Finney, 1984), which also suggest an increase in the NH₃ compensation point in response to biotrophic infection.

Within Leaf Spatial Variability of Apoplast pH and NH₄⁺ Concentration

Apoplast extractions have a number of sources of error. The principal ones have been dealt with above and appear to be quantitatively too small or liable to result in calculated NH₃ compensation points becoming lower. However, it is not possible to ever be completely sure what the origin of the "apoplast" extract really is. The extraction technique has been widely used with a range of validation techniques (Cosgrove and Cleland, 1983; Husted and Schjoerring, 1995) and available data indicate that extracted liquid is apoplastic in origin. A major consideration in the application of this technique to the calculation of NH₃ compensation points is whether or not extracted liquid is representative of liquid in the apoplast at leaf gas exchange sites. Hoffmann and Kosegarten (1995) found spatial differences in the apoplast of leaf cells of sunflower to be up to 1 pH unit. Bowling and Edwards (1984) report differences in apoplast pH of Tradescantia virginiana between guard cell and epidermal cell of 1.4 pH units in the dark, although in the light, gradients were much smaller. However, even these large reported pH gradients are insufficient to account for the largest discrepancies found between NH₃ compensation points determined by the two different approaches (i.e. a ratio of 30). A difference in pH of 0.7 unit is, however, sufficient to account for the mean 5-fold discrepancy. However, the pH of bulk foliar apoplast extracts is unlikely to represent one extreme of spatial apoplast pH variability, but rather some intermediate value.

It is possible that there may be gradients of pH radially through the cell wall due to solute transport and volatilization. The existence of such gradients in plant cell walls is yet to be demonstrated, but such a possibility merits further investigation if a suitable measurement technique could be developed.

NH₄⁺ concentration in the foliar apoplast is unlikely to be spatially uniform, but no investigation of its variation has been carried out to date. Indeed it is doubtful whether a suitable technique for its investigation is currently available. However, reports and speculation on the evaporative concentration of solutes at the end of the transpiration stream such as Canny (1995), would suggest that the apoplast at transpiration sites could be enriched in NH₄⁺ relative to the bulk of the foliar apoplast. Furthermore, such an accumulation of solutes may not be uniform over the leaf but is thought to be greater at leaf margins (Wilson et al., 1991). Consequently extracts of the foliar apoplast would be likely to underestimate the NH₄⁺ concentration at transpiration sites, and thus the NH₃ compensation point.

	CONCLUSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

NH₃ compensation points for L. sylvatica are low compared with values in the literature when determined by either gas exchange measurements or the pH and NH₄⁺ concentration of apoplast extracts. This is consistent with other measurements that suggest that wild species generally have lower NH₃ compensation points than crop plants. The marked discrepancy between compensation point values determined by the two discussed methods is not readily explained, since most conceivable errors are liable to result in overestimates of compensation points, or are of insufficient magnitude. In the absence of other data, spatial variability of pH and/or NH₄⁺ concentration within the foliar apoplast appears the most promising area for further investigation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

Experimental Plants

Wild-growing Luzula sylvatica (Huds.) Gaud. plants were collected from Carlops (Scottish Borders, UK; UK Ordnance Survey grid reference NT179567) and potted singly in a 4:1 vermiculite:perlite mix. Plants were grown in a greenhouse at a minimum temperature of 10°C under ambient light supplemented with mercury vapor lamps during winter months to increase the photoperiod to 16 h day¹. Artificial light intensity was approximately 400 µmol photons m² s¹ at canopy height. Plants received Long-Ashton nutrient solution (Hewitt and Smith, 1974) concentrations of 0.214, 2.14, 5.35, 10.7, or 21.4 mM N as NH₄Cl or NH₄NO₃ at a rate of 20 mL pot¹ week¹ for approximately 8 months prior to use in experiments. This rate of nitrogen addition was equivalent to 2, 20, 50, 100, and 200 kg N ha¹ y¹ based on a growing medium surface area of 156 cm².

During growth, plants were found to be infected with the rust Puccinia luzulae Libert., which is widespread in wild-growing L. sylvatica.

Typical leaf areas ranged from 300 to 1,250 cm² pot¹ depending upon nitrogen treatment.

Gas-Exchange Measurements

Measurements of the exchange of NH₃, water vapor, and carbon dioxide with plants were carried out in a purpose-built dynamic cuvette facility (Fig. 5) contained within a controlled temperature room.

View larger version (52K): [in this window] [in a new window]   Figure 5.   Schematic diagram of gas-exchange measurement system.

Experimental plants were placed inside a 0.216-m³ Perspex cuvette. Pots were placed in polythene bags secured tightly around the base of the foliage with cable ties. Light was supplied with a 250 W discharge lamp at a rate of approximately 400 µmol photons m² s¹ at canopy height after passing through a glass water bath to reduce heating effects. A control experiment was performed, replacing experimental plants with artificial Chlorophytum sp. (Philip Display, Edinburgh) of similar morphology to quantify any gaseous exchange between the growth medium and the cuvette atmosphere. Experiments using artificial plants or an empty cuvette also showed there to be no line loss of ammonia once stable conditions had been reached.

During experiments plants were fed and watered as necessary with a syringe through a 1.6-mm outside diameter Teflon tube sealed inside the plastic bags covering pots.

Air entering (30 L min¹) and leaving the cuvette was continually sampled for NH₃ content with an AMANDA NH₃ analysis system (Wyers et al., 1993; ECN, Petten, The Netherlands) and was sampled for water vapor and carbon dioxide content on a 10-min cycle. Water vapor and carbon dioxide were measured with a temperature and humidity probe (Rotronic Instruments, Horley, Surrey, UK) and an infrared gas analyzer (type 255 mk3, ADC, Hoddesdon, Hertsfordshire, UK), respectively. Air temperature and humidity within the cuvette were continuously monitored using a probe (Vaisala, Cambridge, UK) and leaf temperature was measured using six type-T thermocouples attached to the underside of leaves with paper clips in such a way as to hold the junction against the leaf surface.

Prior to entering the cuvette, the air stream was dried by passing through copper coils submerged in a water bath at 4°C with condensed liquid being captured in a flask. Adjustment of humidity was carried out by bubbling a portion of the air stream through deionized water. Relative humidity was maintained at 50% ± 10%. NO_x was removed by passing air through Purafil and activated charcoal, and NH₃ was removed by passing air through oxalic acid suspended on rockwool. Particles were captured on a depth filter (Pall Biomedical, Portsmouth, Hampshire, UK).

Generation of NH₃ was achieved by introducing, with a peristaltic pump, small volumes of dilute NH₃ solution into a heated flask through which the air stream passed.

Determination of NH₃ Compensation Points

NH₃ compensation points were determined from the linear regression equation of NH₃ flux against ambient NH₃ concentration where the ambient NH₃ concentration was considered to be that of air entering the cuvette. Figure 6 shows a typical plot.

View larger version (17K): [in this window] [in a new window]   Figure 6.   Variation of NH3 flux to and from plant foliage with concentration of ambient NH3. Positive fluxes represent emission from foliage and negative fluxes represent deposition to foliage. Values are means of 11 values from the most stable portions of AMANDA data. Error bars are SEs of 11 values. Plants used in this case received 200 kg N ha1 y1 as ammonium nitrate, but the plot is typical of those used to determine compensation points for all treatments.

Apoplast Extraction

To prepare apoplast extracts, leaves were detached from plants, washed in deionized water, dried with tissues (Kimberly Clark, Aylesford, Kent, UK), and infiltrated with 50 µM indigo carmine solution (4°C) under alternate vacuum and pressure in a 50-mL syringe. Leaves were again dried and rolled perpendicular to the long axis before they were placed in a 15-mL syringe-type serial pipettor tip barrel (Boeringer-Mannheim, Lewes, East Sussex, UK). Pipettor tip and leaves were then placed in a 50-mL centrifuge tube with a 1.5-mL microcentrifuge tube at the bottom to collect liquid expelled from the leaf. The assembly was centrifuged at 2,000g for 4 min at 4°C.

NH₄⁺ and pH Measurement

The pH of extracts collected during centrifugation was measured with an InLab 423 semi-micro electrode (Mettler Toledo, Udorf, Switzerland) inserted in the microcentrifuge tube. Calibration was only with low ionic strength standards (Russell pH, Auchtermuchty, Fife, UK). NH₄⁺ concentration was assessed in an AMFIA NH₄⁺ flow injection analyzer (ECN). Extracts of less than 100 µL were diluted to the minimum analyzable volume of 100 µL with deionized water prior to NH₄⁺ analysis.

Cytoplasmic Contamination

Cytoplasmic contamination of apoplast extracts was assessed by comparison of malate dehydrogenase activity (EC 1.1.1.37) in apoplast extracts and leaf homogenates. Apoplast extracts for assay were prepared as above excepting that a buffer (0.1 M N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid, 2 mM dithiothreitol, and 0.2 mM EDTA) was substituted for indigo carmine solution and leaves were weighed before and after infiltration. Leaf homogenates were prepared by grinding tissue in liquid nitrogen before diluting a weighed portion with the aforementioned buffer. Malate dehydrogenase activity was assessed by adding 30 µL of extract to 1 mL of a solution composed of 0.05 M Tris, 0.1 mM NADH, and 0.4 mM oxalacetate, and measuring absorbance decrease at 340 nm (20°C) in a Shimadzu UV-160A spectrophotometer (Shimadzu, Kyoto).

Assessment of Apoplast Dilution

Dilution of the apoplast liquid during the extraction procedure was assessed by measuring the concentration of indigo carmine in extracts spectrophotometrically at 610 nm. Since dilution was somewhat variable, indigo carmine was utilized in all extracts (excepting those used to assess cytoplasmic contamination) to eliminate errors due to the use of a mean dilution correction, as has been the case in previous studies (e.g. Husted and Schjoerring, 1995). Furthermore, the assessment of apoplast dilution in each sample eliminates interpretation problems resulting from the incomplete infiltration of leaves.

The influence of indigo carmine on pH analysis was tested by twice comparing 10 extracts from leaves infiltrated with deionized water and 10 of those infiltrated with 50 µM indigo carmine.

Standard NH₄⁺ solutions with 0 to 200 µM indigo carmine were analyzed to test for any influence of indigo carmine on NH₄⁺ analysis.

Osmotica

An estimate of the osmolality of the symplast of L. sylvatica was made by using several different methods to produce extracts for analysis in an osmometer (model 5100C vapor pressure osmometer, Wescor, Logan, UT). (a) Leaves were frozen in liquid nitrogen and ground in a pestle and mortar. A weighed portion of the ground leaves was allowed to equilibrate with deionized water overnight in a sealed microcentrifuge tube at 4°C. Following equilibration the sample was centrifuged at 10,000g for 6 min before measuring the osmolality of the supernatant. (b) The above technique was modified by equilibrating ground tissue in deionized water with 1 drop of Triton X-100 50 mL¹ to further disrupt cell membranes. Correction was made for the influence of the Triton X-100 on the osmolality of the extract. (c) A tight roll of leaves was squeezed in a vise and the osmolality of the expelled liquid measured. (d) A roll of leaves was placed in a 5-mL syringe that was sealed with a blocked needle hub. The syringe was placed in liquid nitrogen and liquid was expelled with the syringe plunger after thawing. (d) A variation of the above technique was carried out in which cells were disrupted by boiling rather than freezing. Sealed syringes were immersed in boiling water for 3 min and cooled before expelling liquid from leaves with the syringe plunger.

As a consequence of the widely varying estimates of osmolality resulting from the above methods (see "Results"), osmotica were not used in this study during infiltration. It was considered that because the osmolality of the symplast could not be accurately measured, the effect of infiltrating with osmotica was less predictable than that of infiltrating a well-hydrated leaf with a low osmolality solution, relying on cell turgor to prevent significant transplasmalemma water fluxes.

To test the potential for water to move from the apoplast to the symplast during infiltration with low osmolality solutions, a comparison was made between determinations of the foliar intercellular air space volume when infiltrating with different solutions. Leaves were weighed before and after infiltration with deionized water, high-viscosity silicone fluid (5 cs, Dow-Corning, Poole, Dorset, UK), which theoretically cannot cross the plasmalemma (Cosgrove and Cleland, 1983), and 91 g L¹ D-mannitol solution that was theoretically isoosmotic (see "Results") with the symplast. Account was taken of the density of the infiltrated solutions.

A further comparison was made of the NH₄⁺ concentration of apoplast extracts after infiltration with either 50 or 91 g L¹ D-mannitol solution.

Apoplast Buffering and pH Regulation

To assess the necessity for correction of apoplast hydrogen ion concentration for dilution during the extraction procedure, dilutions with 50 µM indigo carmine were carried out on apoplast extracts while monitoring pH changes.

Further investigation of apoplast pH regulation was carried out by infiltrating leaves from plants receiving 200 kg N ha¹ year¹ as NH₄Cl with 50 µM indigo carmine solution adjusted to pH 3, 4, 7, or 10 with HCl or NaOH. The pH of resulting extracts was compared with the theoretical values based on simple mixing of infiltrated solution and apoplast liquid. To calculate the expected pH of extracts based on simple mixing, it was assumed that the extracts obtained after infiltration with unadjusted indigo carmine solution (pH 7) gave a reasonable estimate of apoplast pH, i.e. that apoplast buffering was sufficient to prevent significant perturbation of apoplast pH during infiltration with a neutral, unbuffered solution.

Regulation of Apoplast NH₄⁺ Concentration

As for the investigation of apoplast pH regulation, leaves from plants receiving the 21.4 mM N NH₄Cl nutrient solution were infiltrated with 50 µM indigo carmine solution adjusted to 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, and 2.0 mg NH₄⁺ L¹ with (NH₄)₂SO₄. Again the NH₄⁺ concentrations of extracts were compared with those predicted from simple mixing, assuming that the extracts obtained following infiltration with unadjusted indigo carmine solution, and correction for dilution, gave a reasonable estimate of apoplast NH₄⁺ concentration.

Calculation of NH₃ Compensation Points

NH₃ compensation points were calculated from apoplast pH and NH₄⁺ concentration according to the equilibria applied by Sutton et al. (1994) using the simplified numerical fit of Nemitz (1998):

(1)

where [NH_3(g)] is the NH₃ compensation point in M, and T is temperature in Kelvin.

The temperature-independent analog for the NH₃ compensation point,  (the ratio of apoplast NH₄⁺ concentration to apoplast H⁺ concentration; Sutton et al.,1996) was also used as a method of comparison.

Ionic Composition of the Apoplast

Apoplast extracts prepared as above were analyzed for inorganic ion content on an Alltech Odyssey high performance ion chromatography system (Alltech, Deerfield, IL) with either an Alltech Universal Cation column (3 mM methane sulfonic acid eluent) or an Alltech Universal anion column (A-15; 0.86 mM Na₂CO₃/0.34 mM NaHCO₃ with 5% [w/v] methanol, eluent). Na⁺ in extracts due to the presence of indigo carmine was subtracted from measured values.

Similar extracts were prepared for organic ion analysis. Extracts were dried in a Heto CT 110 centrifugal drier (Heto-Holten A/S, Allerod, Denmark), dissolved in pyridine, and derivatized in N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide at 70°C for 30 min. Derivatized samples were then analyzed in a VG Organic MD800 gas chromatograph-mass spectrometer (Fisons, Manchester, UK). Quantification of chemical species was carried out by comparison with standards.

A simple computer model based on measured ionic concentrations and organic ion dissociation constants was used to predict the concentration of unquantified negative charge needed to predict a physiologically realistic apoplast pH and the dissociation state of organic ions and NH₄⁺ (Hill, 1999). In the model, changes in pH with increasing dilution were modeled from the concentrations of pH-active species and their dissociation constants at 20°C or 25°C (Weast, 1987; Budavari, 1989) and concentration of inorganic ions. The calculations were derived on the principle of charge balance and the function was solved numerically by iteration using the Newton-Raphson approximation (Burden and Faires, 1993). Concentrations of charged forms of dissociating species were calculated from their dissociation constants, total concentrations, and H⁺ concentrations. The function was of the form:

(2)

Addition of unquantified negative charge was probably necessary due to a greater proportion of positive ions being identified and quantified than negative ions.