Journal of Experimental Botany, Vol. 53, No. 367, pp. 277-286,
February 1, 2002
© 2002 Oxford University Press
Original Papers |
Leaf age-related differences in apoplastic NH4+ concentration, pH and the NH3 compensation point for a wild perennial
1 Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
2 Centre for Ecology and Hydrology, Edinburgh Research Station, Bush Estate, Penicuik, Midlothian EH26 0QB, UK
Received 18 September 2001; Accepted 18 September 2001
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Abstract |
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Extracts of the foliar apoplast of leaves of different ages of Luzula sylvatica (Huds.) Gaud. were prepared by vacuum infiltration and centrifugation. Measurements of pH and
concentration were performed on extracts. From these bioassay measurements the relative magnitude of NH3 compensation points for leaves of different ages were inferred. Young leaves were found to have much higher apoplast pH than old leaves, leading to the calculation of 4–10-fold higher NH3 compensation points. Such age-related differences in the NH3 compensation point are considerably larger than those previously reported. Apoplast pH and concentration were found to increase during leaf expansion before declining prior to senescence. Bulk foliar tissue pH, and total N concentrations were also found to be generally higher in young leaves than in old leaves. Where a significant correlation was found, total foliar N, bulk tissue foliar and the calculated NH3 compensation point were all found to increase with N supplied to roots, whilst apoplast and bulk tissue H+ concentrations were found to decline. The potential of bulk foliar tissue measurements to act as simple predictors of the NH3 compensation point is discussed. Key words: Age-related difference, apoplast, NH3 compensation point, nitrogen, pH.
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Introduction |
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Ammonia is an important atmospheric gas which, when deposited, has various documented detrimental effects on sensitive (low N) semi-natural/natural vegetation and ecosystems, through eutrophication and acidification (Heil and Diemont, 1983
; Alonso and Hartley, 1998; Leith et al., 1999). Detrimental effects on ecosystems generally result in species composition change due to differential use of available N by plant species (Heil and Diemont, 1983), but at very high NH3 concentrations direct toxic effects on vegetation have also been reported (van der Eerden et al., 1992; Thijsse and Baas, 1990; Yin et al., 1998).
Exchange of NH3 with vegetation can be related to the NH3 compensation point (Farquhar et al., 1980; Hill et al., 2001). At atmospheric NH3 concentrations above the NH3 compensation point, NH3 is deposited from the atmosphere to vegetation, whilst at atmospheric NH3 concentrations below the NH3 compensation point, NH3 is emitted from vegetation to the atmosphere.
The principal route of exchange of NH3 with internal plant tissues is through stomata (Sutton et al., 1993). The stomatal NH3 compensation point is determined by the concentration and the pH of the liquid phase of the foliar apoplast at gas exchange sites (Farquhar et al., 1980; Husted and Schjoerring, 1996; Hill et al., 2001). In the last six years attempts have been made to calculate the stomatal NH3 compensation point from measurements of the pH and concentration of extracts of the foliar apoplast (Husted and Schjoerring, 1995). Such extracts have been prepared by infiltrating solutions into leaves before extracting them by centrifugation, a considerably simpler operation than performing lengthy controlled gas exchange measurements, but still a fairly time-consuming process.
It has recently become clear that calculation of the NH3 compensation point from measurements of apoplast extracts frequently leads to substantial underestimates of the NH3 compensation point as compared with empirical measurements of gaseous NH3 exchange (Hill et al., 2001). However, the use of measurements of the pH and concentration of foliar apoplast extracts as a method of comparison of the relative magnitude of the NH3 compensation point in different plants or leaves is not in dispute. Furthermore, calculation of the NH3 compensation point from extracts of the foliar apoplast permits the comparison of classes of leaves rather than entire plants. Such comparisons are not, at present, possible using gas exchange measurements, since these measurements require substantially more plant material to generate NH3 concentration differences of sufficient magnitude to be quantifiable with current state-of-the-art NH3 analysers. Gas exchange measurements carried out on seedlings are not necessarily representative of the young leaves of mature plants.
This study documents substantial differences in the NH3 compensation point, as calculated from extracts of the foliar apoplast, for leaves of different ages in the widespread, wild perennial, Luzula sylvatica. It is the first study to document leaf age-related differences in the NH3 compensation point for a wild plant. At this time the only comparable data concern the crop plant Brassica napus (Husted and Schjoerring, 1995), which was found to exhibit little leaf age-related variation in the stomatal NH3 compensation point.
Data presented by other authors have previously suggested that simple bulk foliar tissue measurements, such as total foliar N and total foliar concentration varied in a similar manner to the NH3 compensation point when plants were subject to differing N nutrition. Discussion is made of the potential of such measurements, to act as predictors of the relative magnitude of NH3 compensation points.
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Materials and methods |
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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 and perlite mix. Plants were grown in a glasshouse at a minimum temperature of 10 °C (maximum temperature varied with ambient) under ambient light supplemented with mercury vapour lamps during the winter months to increase the photoperiod to 16 h d-1. Artificial irradiance was approximately 400 µmol photons m-2 s-1 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 mmol N l-1 nutrient solution as NH4Cl or NH4NO3 at a rate of 20 ml pot-1 week-1 for approximately 8 months prior to use in experiments. This rate of N addition was equivalent to 2, 20, 50, 100, and 200 kg N ha-1 year-1 based on a growing medium surface area of 156 cm2. Attempts to grow L. sylvatica with 
as the sole N source resulted in stunted, chlorotic plants, and were consequently abandoned. Additional watering was carried out with deionized water. During growth, plants were found to be infected with the rust Puccinia luzulae Libert. which is widespread in wild-growing Luzula sylvatica.
Apoplast extraction
In order to prepare apoplast extracts, leaves were detached from plants (maintained at approximately 20 °C), washed in deionized water, dried with tissues (Kimberly Clark, Aylesford, Kent, UK), and infiltrated with 50 µmol l-1 indigo carmine solution (4 °C) under alternate vacuum and pressure in a 50 ml syringe. All plants were well hydrated prior to infiltration to avoid significant transplasmalemma fluxes of water. Comparison of extracts obtained following infiltration with different concentrations of D-mannitol did not show any significant effect on the pH or concentration of extracts or on the infiltration volume. Leaves were again blotted dry, and rolled perpendicular to the long axis before placing 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 2000 g for 4 min at 4 °C. The time lapse between detaching leaves from the plant and centrifugation was approximately 5 min. Extractions were not carried out on plants within 24 h of N feeding.
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 (conductivity c. 0.4 mS cm-1) (Russell pH, Auchtermuchty, Fife, UK).
concentration was assessed in an AMFIA flow injection analyser (ECN, Petten, Netherlands) (detection limit 0.6 µM). Extracts of less than 100 µl were diluted to the minimum analysable volume of 100 µl with deionized water prior to analysis. This was a 50:50 dilution in most cases.
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 mol l-1 N-TRIS[hydroxymethyl]methyl-2-aminoethanesulphonic acid, 2 mmol l-1 dithiothreitol and 0.2 mmol l-1 EDTA) was substituted for indigo carmine solution, and leaves were weighed before and after infiltration. Leaf homogenates were prepared by grinding tissue in liquid N2 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 mol l-1 TRIS, 0.1 mmol l-1 NADH and 0.4 mmol l-1 oxalacetate, and measuring absorbance decrease at 340 nm (20 °C) in a Shimadzu UV-160A spectrophotometer (Shimadzu, Kyoto, Japan).
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 and interpretation problems resulting from the incomplete infiltration of leaves. Tests were carried out to ensure that the presence of indigo carmine in the infiltration solution did not influence the measurement of pH and concentration (Hill et al., 2001).
Apoplast extracts of young and old leaves
Apoplast extracts were collected from young and old leaves of plants from all N treatments after infiltration with 50 µM indigo carmine solution. Young leaves were considered to be those from closest to the centre of the plant up to and including the youngest fully expanded leaves. Old leaves were the oldest leaves not showing visible signs of senescence from the outer portions of the plant. It was not possible to number the leaves when comparing leaves from different N treatments, since the total number of leaves varied considerably between treatments. Variation in leaf size also enforced the use of varying numbers of leaves in each extraction. After preparation, extracts were analysed for pH and concentration as above.
Apoplast extracts from leaves within different age classes
Extracts were also prepared as above for four age classes of leaves from the treatment receiving 200 kg N ha-1 year-1 NH4NO3 nutrient solution. Ignoring the first two very small leaves at the centre of the plant, leaves were counted from the centre of the plant into the classes 1–3, 4–6, 7–9, and 10+. For plants from this treatment the 1–3 and 4–6 and 7–9 classes approximately corresponded to the young age class used above, and the 10+ to the old. Extracts were analysed for pH and concentration by the aforementioned methods.
Calculation of the NH3 compensation point
NH3 compensation points were calculated from apoplast pH and concentration according to the equilibria applied by Sutton et al. (Sutton et al., 1994) using the simplified numerical fit of Nemitz et al. (Nemitz et al., 2000):
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Bulk foliar concentration of young and old leaves
Five young and five old leaves were selected using the same criteria as for apoplast extractions. After washing and drying as for the apoplast extracts, leaves or leaf sections, were frozen in liquid N2 and ground in a pestle and mortar. Portions of the ground tissue were placed into weighed microcentrifuge tubes, which were then reweighed. One ml of deionized water was added to each tube and samples were used for pH measurement, frozen and stored at -20 °C until analysis. Immediately prior to analysis, in the AMFIA analyser, samples were thawed and centrifuged at 15000 g for 1 min. The supernatant was filtered under pressure through a small plug of cotton wool in a 1 ml pipette tip. Prior to use the cotton wool was washed in deionized water before rinsing in approximately 200 µl of extract. Pressure for filtration was generated with a small air pump. Values of concentration were corrected for dilution during extraction.
Total N concentration of leaves of different ages
One leaf of each age (counting from the centre of the plant) was detached from each of three plants in the 200 kg N ha-1 year-1 NH4Cl treatment. These were washed in deionized water and dried in an oven at 80 °C until there was no further weight change. After drying each group of three leaves was ground in a ball mill, wrapped in tin capsules and analysed for N concentration in a Carlo Erba Model 1106 Elemental Analyser (Carlo Erba, Milan, Italy) calibrated with an atropine standard.
Using the same selection criteria as for apoplast extracts and bulk tissue measurements, samples were prepared as above for young and old leaves of all nitrogen treatments.
Photosynthesis and stomatal conductance of leaves from different age classes
Measurements of photosynthetic rate and stomatal conductance were carried out on individual young and old leaves from plants from four N treatments (200 and 50 kg N ha-1 year-1 as NH4Cl and NH4NO3) using a Li-6200 Portable Photosynthesis System (Li-Cor, Lincoln, Nebraska, USA).
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Results |
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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.
Apoplast pH and concentration of young and old leaves
It is clear (Fig. 1) that old leaves had a consistently lower apoplast pH and calculated NH3 compensation point than young leaves. pH differences were statistically significant (t-test assuming unequal variance, Microsoft Excel 97) in eight of the ten treatments. Calculated NH3 compensation point differences were statistically significant in six of the ten treatments. Apoplast concentrations did not show a leaf age-related trend on the basis of comparing young and old leaves.
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0.05) between young and old leaves.No significant relationship between nitrogen supply to roots and apoplast H+ concentration, apoplast
concentration or the NH3 compensation point was found, excepting in measurements carried out on old leaves of plants receiving NH4NO3, where all apoplast measurements gave significant regressions against root nitrogen supply (Fig. 2)
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Apoplast pH and concentration of leaves from different age classes
Apoplast pH, apoplast concentration and the calculated NH3 compensation point (Fig. 3) all showed an increase with the age class of leaves assayed before decreasing in the ‘10+’ class.
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Bulk foliar concentration and pH
Both the concentration and pH (Fig. 4) of bulk foliar tissue extracts were consistently lower in old leaves than in young leaves. Age-related concentration differences were statistically significant in four of the ten treatments, and pH differences in seven of the ten treatments.
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Young leaves receiving either N form showed a significant decline in bulk tissue H+ concentration with N supplied to roots (Fig. 5
). Bulk tissue increased significantly with N supply in all treatments excepting old leaves of plants receiving N as NH4Cl (Fig. 6).
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Total foliar N concentration
A statistically significant increase in total foliar N concentration with root N supply was found for young and old leaves receiving N as NH4Cl or NH4NO3 (Fig. 7). No significant difference was found between young and old leaves for either N treatment (data not shown). However, when measurements were carried out on leaves of all ages rather than young and old classes, the total N concentration of leaves initially increased with age (Fig. 8) before gradually declining to the oldest leaves.
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Effect of form of N supplied to roots
No consistent, statistically significant differences between plants receiving N as NH4Cl and those receiving N as NH4NO3 were found for any of the measured parameters.
Photosynthesis and stomatal conductance
No significant differences in photosynthetic rate or stomatal conductance were found between young and old leaves, or in relation to N form or N supply. (Mean values of carbon assimilation rate and stomatal conductance for leaves of all ages and N treatments were 2.8±0.1 µmol m-2 s-1 and 63±5 mmol m-2 s-1, respectively.)
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Discussion |
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Given the lack of age-related differences, for the young and old leaf comparison, in apoplast
concentration and the consistently lower pH of the apoplast of old leaves (Fig. 1
), the lower calculated NH3 compensation points of old leaves appear to be pH-driven. The mean 4-fold difference in NH3 compensation point between young and old leaves was due to a mean 4-fold difference in apoplast H+ concentration.
Somewhat in contrast to the aforementioned findings, trends of apoplast pH, apoplast concentration, and the calculated NH3 compensation point are visible in Fig. 3 where four leaf age classes were compared. These data show that there was an increase in the apoplast pH and concentration with leaf age as the young leaf expanded, followed by a decrease in both prior to senescence. In this case the changes in NH3 compensation point were driven by both apoplast pH, and apoplast concentration. However, whilst the difference in apoplast concentration between the ‘7–9’ and ‘10+’ age classes (0.032–0.026 mmol l-1) gave rise to a 1.2-fold drop in NH3 compensation point, the pH difference between the two age classes (6.63–5.41) gave rise to a 17-fold drop in the NH3 compensation point. Thus, the combined effect on the NH3 compensation point (a 20-fold drop between the ‘7–9’ and ‘10+’ age classes) was dominated by the difference in apoplast pH between the leaf age classes.
The two sets of measurements carried out on the apoplast with respect to leaf age are broadly consistent, since the ‘young’ leaves of Fig. 1 approximately correspond to the ‘1–3’, ‘4–6’ and ‘7–9’ age classes in Fig. 3, and the ‘old’ leaves correspond to the ‘10+’. If the ‘1–3’, ‘4–6’ and ‘7–9’ age classes are considered as one young age class, the young class has an apoplast concentration 0.003 mmol l-1 lower than that of the old age class. Thus, the 10-fold lower NH3 compensation point of the old age class, resulting from this treatment of the data, is entirely due to the pH difference between the young and old leaves (6.38–5.32, respectively).
Due to the morphological differences between B. napus and L. sylvatica, it is not possible to make much meaningful comparison between the variation in apoplast and pH with leaf age presented here, and that presented earlier (Husted and Schjoerring, 1995). However, given the lack of other measurements of both apoplast concentration and apoplast pH for leaves of different ages in the literature, some comparison is in order. In marked contrast to this study they found no change in apoplast pH with leaf age, but possibly in line with the data presented in Fig. 3, they found the apoplast concentration to be significantly higher in young leaves. The maximum difference in NH3 compensation point between young and old leaves found by Husted and Schjoerring due to the difference in apoplast concentration was only 1.4-fold (Husted and Schjoerring, 1995). This figure is considerably less than age-related NH3 compensation point differences found for L. sylvatica, but similar to the difference in NH3 compensation point due to the concentration difference found between leaves from the ‘7–9’ and ‘10+’ leaf age classes.
Effects of infiltration and fungal infection on the pH of the foliar apoplast
There are reports of exposure to fungal elicitors leading to extracellular alkalization in plant cell cultures (Fukuda, 1996; Tripathy et al., 1999). As indicated in ‘Materials and methods’, a biotrophic fungal infection was widespread in the experimental material; such infections are known to cause apoplastic alkalization (Tetlow and Farrar, 1993; Fukuda, 1996). Using the same technique as that employed here to extract the foliar apoplast, Tetlow and Farrar found a 5-fold decrease in apoplastic H+ activity in rust-infected leaves of barley relative to uninfected leaves (Tetlow and Farrar, 1993). Since older leaves of Luzula tended to have a greater number of pustules, it would be expected that older leaves would have a higher apoplastic pH as a result of fungal infection. Since, by contrast, older Luzula leaves had lower apoplastic pH values than did younger leaves, it is possible that the apoplast of older leaves would have been even more acid relative to the apoplast of younger leaves than was observed here if there had been no fungal infection.
No direct comparisons were made of cytoplasmic contamination as a function of leaf age; material for contamination assays was collected across the age range. However, Husted and Schjoerring, using the same techniques as those employed here, reported similar values (<1%) of cytoplasmic contamination of the apoplast of leaves of oilseed rape, but did not find any age-related effects (Husted and Schjoerring, 1995).
Stoichiometric considerations of processes which could alter apoplastic and bulk pH
Metabolism involving net production of protons: The observed decrease in pH of both apoplast and bulk leaf with increasing leaf age is consistent with the conversion of a weak acid (CO2) and a neutral compound (sugar) into a strong organic acid (e.g. malic acid) (Smith and Raven, 1979). Acid–base regulation in the cytoplasm could lead to decreases in apoplastic and vacuolar pH, with appropriate fluxes of co- or counter-ions (e.g. malate) (Walker, 1976; Smith and Raven, 1979; Ullrich, 1992). A similar acidification of both vacuole and apoplast could result from the assimilation in the cytoplasm of older leaves of a fraction of the taken up by the roots, with transfer of the resulting near-neutral organic nitrogen to younger leaves and other growing organs (Smith and Raven, 1979; Hill et al., 2001). Detailed investigation of the composition of plant parts (including xylem sap) as a function of plant age are needed to examine these hypotheses as to the cause of the observed changes in apoplast and bulk leaf pH.
Transport processes involving recycling of H+ across membranes:
Active transport of solutes such as sugars (Malek and Baker, 1977; Bush, 1993; Sauer et al., 1994) and phosphate (Sakano, 1990) across the plasmalemma of plant cells is usually energized by an H+ electrochemical potential difference generated by a P-type ATPase. Such transport may increase, on a leaf weight basis, during apoplastic loading of scavenged materials into the phloem in older leaves prior to senescence (noting that there seems to be no evidence as to the apoplastic or symplastic nature of phloem loading in the Juncaceae: Giaquinta, 1983; Eschrich and Fromm, 1994; Opaskornkul et al., 1994). However, there are no sound a priori grounds for assuming a significant effect on apoplast pH of a change in the rate of such H+-coupled transport processes (Walker, 1976; Smith and Raven, 1979; Ullrich, 1992). Furthermore, any decrease in apoplast pH related to H+-coupled transport would, in the absence of other metabolic processes influencing pH, increase the pH of the cytoplasm, and consideration of the relative volumes and buffer capacities of the intracellular compartments and the apoplast (Winter et al., 1993; Yin et al., 1996) mean that the bulk leaf pH would not necessarily fall during any such cotransport-related apoplast acidification.
These arguments suggest that variations in the rate of H+-cotransport processes are relatively unlikely to explain the observed decrease in apoplast and bulk leaf pH with increasing leaf age.
The acid growth hypothesis:
Apoplast pH change with respect to leaf age appears to be considered in the literature only in relation to the ‘acid growth hypothesis’ (Hager et al., 1971; Taiz, 1984), which suggests that acidification of cell walls, probably due to organic acid synthesis, increases their plasticity and thus allowing expansion of the leaf. Whilst the exact mode of action of auxin in inducing growth remains a matter of debate (Kutschera and Schopfer, 1985; Peters et al., 1998), there appears to be good evidence that the apoplast undergoes acidification during growth (Grignon and Sentenac, 1991; Lüthen et al., 1990; Peters et al., 1998). In the light of this, it is somewhat surprising that the oldest non-senescent leaves of L. sylvatica consistently had a lower apoplast pH than younger leaves (Figs 1, 3). There is, however, some support for the acidification of the apoplast during growth from the observation of a gradual increase in pH with leaf age shown in Fig. 3. The dramatic drop in pH in the oldest measured leaves must to be due to some other process.
Photosynthesis:
A lower photosynthetic rate in old leaves might also offer some explanation of the lower apoplast pH since the apoplast has been found to become alkalinized during photosynthesis (Raven and Farquhar, 1989). Measurements found no significant difference in the photosynthetic rate of young and old leaves, and as with solute transport, the expected corresponding acidification of the symplast was not apparent in bulk extracts. The lack of response of bulk extracts could, however, be due to intracellular buffering, H+-consuming processes, for example, net phosphoglycerate reduction and/or differences in volumes of cell compartments.
There has also been a report of apoplast acidification during photosynthesis (Hanstein and Felle, 1999), but the same arguments against this being a cause of leaf age-related apoplast pH differences apply.
Response of apoplast pH, apoplast concentration and the calculated NH3 compensation point to N nutrition
In contrast to the response of the NH3 compensation point to variations in leaf age, which was dominated by changes in apoplast pH, the response of the NH3 compensation point to variations in N supply (Fig. 2), where a statistically significant relationship was found, was driven almost equally by both apoplast concentration and apoplast pH. The approximately 10-fold mean increase in the calculated NH3 compensation point between the 2 and 200 kg N ha-1 year-1 treatments for old leaves of plants receiving N as NH4NO3 (Fig. 2) was due to a mean 2.6-fold increase in apoplast concentration and a mean 2.9-fold decrease in apoplast H+ concentration. The data of Mattsson et al. for barley show a statistically significant linear relationship between root N supply and the NH3 compensation point for nutrient solution concentrations between 0.5 and 5 mM, with a relatively smaller increase in the NH3 compensation point between the 5 mM and 10 mM in the nutrient solution (Mattsson et al., 1998). Where the response to N supply was linear, a 26-fold increase in the NH3 compensation point was due to a 38-fold increase in apoplast concentration, the effect of which on the NH3 compensation point was reduced by a 1.4-fold increase in apoplast H+ concentration. Their data for oilseed rape also show that changes in the NH3 compensation point with increasing N supply (12-fold increase) were dominated by a 7-fold increase in apoplast concentration, with only a 1.7-fold decrease in apoplast H+ concentration.
Bulk foliar pH, concentration and total N
Direct comparison of bulk foliar H+ concentration, bulk foliar concentration or total foliar N and the calculated NH3 compensation point for L. sylvatica did not show a statistically significant relationship in this investigation, although the response to both leaf age (Figs 1, 3, 44, 8) and root N supply (Figs 2, 5, 6, 7) was in the same direction for both bulk tissue and apoplast measurements. The data of Husted and Schjoerring for oilseed rape show a statistically significant linear relationship between bulk foliar concentration and the calculated NH3 compensation point for different root N supply (Husted and Schjoerring, 1996). Schjoerring et al. present data showing a statistically significant linear relationship between both bulk foliar N and bulk foliar and the NH3 compensation point for oilseed rape (Schjoerring et al., 1998). The data of Mattsson et al. for barley also show a statistically significant linear relationship between bulk foliar concentration and the NH3 compensation point (Matsson et al., 1998). Given these good relationships between bulk foliar measurements and apoplast measurements in the literature, simple bulk tissue measurements would seem to have some potential as indicators of the NH3 compensation point. However, since no significant correlation was found between bulk and apoplast measurements for L. sylvatica, the relationship between them would need to be established for the individual plant species under investigation before bulk measurements could be used as indicators for the NH3 compensation point.
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Conclusion |
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There appears to be no obvious explanation for the acidification of the apoplast and bulk tissue, in old leaves of Luzula sylvatica relative to young leaves.
Despite the difficulty in determining a plausible reason, the fact remains that young leaves of L. sylvatica had a consistently higher foliar apoplast and bulk foliar pH than older leaves. There is also evidence to suggest that foliar apoplast concentration increases during leaf expansion before declining prior to senescence. These changes in apoplast pH and concentration give rise to higher NH3 compensation points for young leaves than for old. Young leaves, therefore, have a greater potential for loss, and a lower potential for gain, of N as gaseous NH3. Such dramatic age-related differences in the NH3 compensation point, in the one perennial investigated, make it clear that care must be taken when inferring NH3 compensation points from bioassays, since there may be considerable variation within individual plants. The markedly lower apoplast pH, and consequent very low NH3 compensation point, of older leaves relative to younger leaves merits further investigation to determine the cause of such striking leaf age-related variation.
A significant correlation was not found between apoplast measurements and bulk measurements for Luzula sylvatica in this investigation. Thus, despite the statistically significant correlations which can be seen in published data for other plants, any use of bulk measurements as indicators of the NH3 compensation point would require validation for the plant species under investigation.
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Acknowledgments |
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This investigation was supported by a CASE studentship (ref. GT4/95/64/T) from the Natural Environment Research Council, and funds from the UK Department of Environment Transport and Regions (UMBRELLA project). MA Sutton is grateful to the EC for funding under the GRAMINAE programme (ENV4-CT98-0722). Thanks also to Robert Hardy for allowing experimental plants to be gathered from his land and Frank Harvey and Colin McBeath for gathering experimental plants.
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Notes |
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3 Present address and to whom correspondence should be sent: Institute of Environmental Science, University of Wales, Bangor, Gwynedd LL57 2UW, UK. Fax: +44(0)1248 383646. E-mail: p.w.hill{at}bangor.ac.uk
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