Journal of Experimental Botany, Vol. 53, No. 379, pp. 2441-2450,
December 1, 2002
© 2002 Oxford University Press
Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate
Received 30 March 2002; Accepted 31 July 2002
Department of Plant Ecology, Institute of Biological Sciences, University of Aarhus, Nordlandsvej 68, 8240 Risskov, Denmark
1 To whom correspondence should be addressed. Fax: +45 8942 4747. E-mail: Hans.Brix{at}biology.au.dk
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Abstract |
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The NH4+ and NO3– uptake kinetics by Typha latifolia L. were studied after prolonged hydroponics growth at constant pH 3.5, 5.0, 6.5 or 7.0 and with NH4+ or NO3– as the sole N-source. In addition, the effects of pH and N source on H+ extrusion and adenine nucleotide content were examined. Typha latifolia was able to grow with both N sources at near neutral pH levels, but the plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and higher affinity for uptake of inorganic nitrogen when grown on NH4+. Growth almost completely stopped at pH 3.5, irrespective of N source, probably as a consequence of pH effects on plasma membrane integrity and H+ influx into the root cells. Tissue concentrations of the major nutrients and adenine nucleotides were severely reduced at low pH, and the uptake capacity for inorganic nitrogen was low, and more so for NO3–-fed than for NH4+-fed plants. The maximum uptake rate, Vmax, was highest for NH4+ at pH 6.5 (30.9 µmol h–1 g–1 root dry weight) and for NO3– at pH 5.0 (31.7 µmol h–1 g–1 root dry weight), and less than 10% of these values at pH 3.5. The affinity for uptake as estimated by the half saturation constant, K
, was lowest at low pH for NH4+ and at high pH for NO3–. The changes in Vmax and K were thus consistent with the theory of increasing competition between cations and H+ at low pH and between anions and OH– at high pH. Cmin was independent of pH, but slightly higher for NO3– than for NH4+ (Cmin(NH4+) 
0.8 mmol m–3; Cmin(NO3–) 2.8 mmol m–3). The growth inhibition at low pH was probably due to a reduced nutrient uptake and a consequential limitation of growth by nutrient stress. Typha latifolia seems to be well adapted to growth in wetland soils where NH4+ is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant’s ability to modify pH-conditions in the rhizosphere. Key words: Adenine nucleotides, ammonium, cattail, H+ extrusion, Michaelis-Menten, nitrate, pH, Typha latifolia, Typhaceae, uptake kinetics.
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Introduction |
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Higher plants are able to take up and assimilate nitrogen as NH4+, NO3– and various soluble organic compounds such as urea (CO(NH2)2) and amino-acids (Marschner, 1995; Falkengren-Grerup et al., 2000). In aerated soils with a pH above 4, NO3– is the prevailing N compound and NH4+ is found only in low concentrations, but in waterlogged soils the ratio between NO3– and NH4+ is reversed mainly as a consequence of depressed bacterial nitrification activity and denitrification of NO3– (Armstrong, 1982). Ammonium is taken up by plant roots by carrier-mediated diffusion of NH4+ across the plasma membrane (Ninnemann et al., 1994) and, at high pH, by free diffusion of NH3 and NH4OH (Kleiner, 1981). Nitrate is taken up by an H+-cotransport system in the plasma membrane (Mistrik and Ullrich, 1996). Uptake and assimilation of NH4+ is a proton-generating process and usually leads to a decrease in the external pH and in the contents of carboxylates in the roots, whereas NO3– uptake and assimilation is a proton-consuming process and usually leads to an increase in external pH and in the contents of carboxylates in the roots (Marschner, 1995). The cytoplasmic pH must, however, be kept fairly constant at or near neutral pH, independently of the external pH, by biophysical and biochemical pH-stat mechanisms in order not to disrupt the cellular processes which are sensitive to pH (Raven and Smith, 1976; Raven, 1986; Gerendas and Schurr, 1999).
The uptake of NH4+ generally decreases with decreasing external pH, probably as a result of increasing competition between H+ and NH4+ for sites on plasma membrane carriers at low pH and an increasing proportion of NH3 and NH4OH at high pH (Kleiner, 1981; Dyhr-Jensen and Brix, 1996). By contrast, the uptake of NO3– is largely unaffected or may even increase at slightly acid pH levels because of the higher H+ gradient across the plasma membrane at low pH, and possibly because increased H+ influx reduce the membrane potential and facilitate NO3– uptake (Marcus-Wyner, 1983; Vessey et al., 1990).
Plant species with a preference for NH4+ are usually found growing in low temperature climates or in acid soils where NH4+ is the prevailing inorganic N source, whereas species with NO3– preference are growing in alkaline soils with a high Ca content (Gigon and Rorison, 1972). The nitrogen preference of higher plants, therefore, appears to be associated with the prevailing inorganic nitrogen form at the natural habitat of the plant. From this, it could be expected that wetland plants, which grow in waterlogged soils where NH4+ is the dominant form of inorganic N, will have a preference for NH4+ over NO3–. Growth with NH4+ as the sole N source may, however, be problematic for the plants compared to growth with NO3– or mixed N sources. Many plant species have reduced growth if fed exclusively with NH4+, and the plants develop a number of characteristics associated with NH4+ toxicity, especially at low root medium pH.
The ability to grow with NH4+ as the sole N-source may be affected by the pH tolerance of the plants. For some terrestrial species intolerant of low pH in the root medium, the net H+ release by the plasma membrane proton pumps are reduced at low external pH, probably because of inadequate energy supply to the roots (Schubert et al., 1990). The plants thus become unable to maintain the electrochemical potential gradient necessary for ion uptake, as the membrane is partially or completely depolarized. Some species may be able to cope with high H+ activity in the root medium by increasing root respiration rate and ATP concentrations, which in turn increases the activity of H+ATPases in the plasma membrane and thus the H+-extrusion (Yan et al., 1992). The difference between tolerant and non-tolerant species could, therefore, be an unequal ability to maintain adequate energy supply to the electrogenic proton pumps in the root plasma membranes.
Several species of wetland plants grow on the margins of lakes with an open water pH of 3 and below and are obviously exposed to extremely acidic conditions around the roots (Fyson, 2000). Species like Typha latifolia L. occur in waters receiving acid mine drainage with a pH of 3.4–3.5, and must therefore possess a tolerance or an avoidance strategy towards NH4+ accumulation and low pH in combination (Schuurkes et al., 1986; Wieder et al., 1990). Whether wetland plants in general are tolerant and able to overcome the detrimental effects of NH4+ nutrition are not yet evident, since most studies have been made with crop species. Rice (Oryza sativa L.) avoids accumulation of NH4+ in the tissue by having a higher assimilation capacity for NH4+ than species adapted for growth with NO3– (Magalhaes and Huber, 1991).
In an earlier study it was demonstrated that T. latifolia was able to grow in solution culture with NH4+ as the sole N-source and to withstand a low medium pH for a few days (Dyhr-Jensen and Brix, 1996). With prolonged exposure (weeks) to pH 3.5, however, the plants showed severe symptoms of stress and stopped growing. The objectives of the present investigation were to elucidate if the form of inorganic nitrogen supplied to the plants, NH4+ versus NO3–, affected their growth and pH tolerance, and to estimate NH4+ and NO3– uptake kinetics as influenced by root medium pH. Furthermore, the proton balance, root respiration, and nutrient and adenine nucleotide contents in the plant tissues were examined in order to identify plant physiological responses.
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Plant material
Shoots of Typha latifolia L. were collected at lake Brabrand, a natural wetland in Denmark (56°10' N, 10°10' E) during July. The plants were rinsed with tap water and propagated in aerated nutrient solutions in 30 l vessels in a growth cabinet (Weiss Umwelttechnic, Lindensruth, Germany) at 20 °C, 85% relative air humidity (RH) and a light:dark cycle of 16:8 h. The photon flux density (PFD) in the growth chamber was approximately 450 µmol m–2 s–1 PAR at the top of the plants. The composition of the nutrient solution was (µmol l–1): NH4+ 1000; SO42– 696; Mg2+ 375; Ca2+ 250; Si(OH)4 125; K+ 313; PO43– 200; Cl– 13; BO33– 6; Mn2+ 0.5; Zn2+ 0.5; Cu2+ 0.1, and MoO42– 0.1. Iron (Fe2+) was added as FeSO4 each day. The pH was adjusted to 6.0 and the nutrient solutions were replaced twice every week to avoid significant changes in pH and depletion of nutrients. After approximately 2 weeks when new roots and leaves had developed, 64 plants of uniform size (approximately 30 cm height) were selected from the stock of plants and mounted in the growth units of the PNT for the NH4+ experiment (see below). The NO3– experiment was initiated 6 weeks later, and plants of a similar size were propagated as described above by separating ramets from the primary shoots. Senescent plant material was removed before the plants were mounted in the PNT.
Experimental facility
The experiments were carried out in the Phyto-Nutri-Tron (PNT); a computer-controlled hydroponics system for whole-plant ecophysiological studies (Lorenzen et al., 1998). The PNT consists of four separate growth units, each with eight root vessels positioned in a block design in a temperature, humidity and light-controlled growth cabinet (Weiss Umwelttechnik, Lindenstruth, Germany). The four growth units were each connected to aerated and temperature-controlled nutrient supply units (180 l) through which the nutrient solutions were recirculated at a rate of approximately 25 l min–1 (3.0 l min–1 per root chamber). The nutrient solutions were UV-sterilized and the pH of the solutions were controlled by a pH-stat system (Knick 2-way PI pH-stat; ±0.2 pH-unit) adding 10 or 100 mM NaOH and 10 or 100 mM H2SO4 (concentration depending on nitrogen source). The air and nutrient solution temperatures were kept at 20±0.2 °C, the RH at 85% and the PFD at 450 µmol m–2 s–1 PAR at the top of the plants with a light:dark cycle of 16:8 h. The composition of the nutrient solutions were (µmol l–1): NH4+ or NO3– 100; SO42– 75; Mg2+ 38; Ca2+ 32; Si(OH)4 13; K+ 27; PO43– 12; Cl– 5; BO33– 3; Mn2+ 0.2; Zn2+ 0.2; Cu2+ 0.05; MoO42– 0.05; Fe2+ 2.5. The nutrient concentrations were maintained nearly constant by a computerized feedback system controlling peristaltic pumps for the delivery of nutrient stock solutions. The concentrations of phosphate in the nutrient solutions were analysed with an autoanalyser assembly every 12 min, and all nutrients were supplied continuously at a rate equivalent to the rate of phosphorus uptake from solution. The NH4+ or NO3– concentrations in the nutrient solutions were monitored daily by flow injection analysis (Lachat Instr., Millwaukee, USA) and adjusted (if necessary) to the desired set point (100 µmol l–1). Intermittent renewal of a third of the nutrient solutions ensured that the concentrations of the major nutrients were maintained within ±10% of the desired set point. Each root vessel (700 mm, diameter 80 mm) had a lid with two openings for the mounting of plants, i.e. a total of 16 plants per treatment were used. Net H+-release during growth with NH4+ or NO3– was estimated from volumes of NaOH and H2SO4 added to maintain a constant pH in the nutrient solutions and related to uptake of NH4+ and NO3–, respectively (Allen and Allen, 1987)
Ammonium uptake kinetics experiments
The plants were grown with (NH4)2SO4 as the sole N source for 6 weeks and with the acidity of the nutrient solutions controlled at pH 3.5, 5.0, 6.5, and 7.0 in the four growth units of the PNT. After 4 weeks the NH4+ uptake kinetics of individual plants from each treatment were estimated by the depletion technique (Brix et al., 1994). Each plant (n=4) was held in a separate 0.5 l root vessel placed in a growth chamber (Weiss Umwelttechnik, Lindenstruth, Germany) with temperature and light conditions similar to those of the PNT. The root vessels were protected from the light and placed in a 20 °C water reservoir for temperature control. The nutrient solution in the root vessel was stirred by a magnetic stirrer and aerated continuously to ensure mixing. The nutrient solution was similar to the solution used in the PNT, except for the initial NH4+ concentration, which was set at 50 µmol l–1 at the initiation of the depletion studies The NH4+ concentration of the nutrient solution was analysed continuously during depletion with a flow colorimeter (Chemlab Instruments Ltd., Essex, UK) at 660 nm. The pH was maintained constant by a pH-stat system (Knick 2-way PI pH-stat; ±0.2 pH unit). After the uptake studies, the plants were divided into roots, rhizomes and leaves and dried at 105 °C. The NH4+ uptake was related to root dry weight.
Nitrate uptake kinetics experiments
Sixty-four plants propagated as described above were transferred to the PNT and grown for 6 weeks with KNO3 as the only N source and with the acidity of the nutrient solutions controlled at pH 3.5, 5.0, 6.5, and 7.0 in the four growth units of the PNT. As the plants were propagated with ammonium as the nitrogen source (see above), the initial 2 weeks were regarded as an acclimation period to nitrate. After 4 weeks the NO3– uptake kinetics were estimated. Since pilot experiments showed that handling and moving of plants to single root vessels in another growth cabinet affected NO3– uptake for a significant period of time, the NO3– uptake kinetics were estimated using the depletion technique on plants in individual root vessels within the PNT (n=4). This minimized the disturbance of the plants, and the NO3– uptake was apparently not affected by the handling. The nutrient solution was similar to the solution used in the PNT, except for the initial NO3– concentration, which was set at 50 µmol l–1 at the initiation of the depletion studies. During depletion the NO3– concentration in the nutrient solution was analysed colorimetrically at 540 nm by flow injection analysis after reduction to NO2– (Lachat Instr., Millwaukee, USA). The pH was maintained constant by a pH-stat system (Knick 2-way PI pH-stat; ±0.2 pH unit). After the uptake study, the plants were divided into roots, rhizomes and shoots and dried at 105 °C. The NO3– uptake was related to root dry weight.
Plant growth and mineral composition
Fresh weights of all plants were determined initially, twice during the experiment (after 2 weeks and 4 weeks), and at the end of the experiment (after 6 weeks) by a standardized weighing procedure. At the set up of the experiments eight plants were divided into roots, rhizomes and shoots, and dried to constant weight at 105 °C to estimate shoot to root and fresh to dry weight ratios. At the end of the experiments all plants were divided, weighed, washed in distilled water and then dried in a forced ventilated oven at 105 °C for dry weight determination. The fresh weights and the average fresh to dry weight ratios were used to calculate the initial dry weight of each individual plant. Relative growth rates (RGR) were calculated for each plant as: RGR=(lnWf–lnWi) t–1 where Wf and Wi are the final and initial total dry weight of the plant and t the period in days. The chemical composition of shoots, roots and rhizomes of plants harvested initially and at the end of the experiments was analysed. The dried plant material was ground and analysed for Kjeldahl-N using a standard procedure (Kjeltex auto 1030 analyser, Tecator, Sweden). The contents of major elements (P, K, Ca, Mg, S, Na) and trace elements (Fe, Mn, Zn, Cu, Si, Mo, B) were analysed by plasma emission spectrometry (ICP-AES, Plasma 2000, Perkin Elmer, USA) after digestion of ground material in HNO3-H2O2 (Brix et al., 1983).
Adenine nucleotides
Pieces of leaves and fresh roots were sampled, immediately placed in small plastic bags containing approximately 20 ml of distilled water, and frozen in liquid nitrogen within 30 s of initial harvesting from plants (Mendelssohn and McKee, 1985). The frozen samples were then freeze-dried, cut into small pieces by a razor blade, and subsamples were analysed for adenine nucleotides according to the method described by Mendelssohn and McKee (1981). Several authors have independently confirmed that this extraction method gives high yields and recoveries of adenine nucleotides from plant tissues (Guinn and Eidenbock, 1972; Mendelssohn and McKee, 1981; Delistraty and Hershner, 1983). Adenosine mono-, di- and triphosphates were measured using the ATP-dependent light yielding reaction of the firefly-lantern luciferin luceferase complex (FLE-50, Sigma Chemical Co.) with a Model LS7500 Beckman liquid scintillation counter. ATP was measured directly; ADP and AMP were converted enzymatically to ATP and determined by subtraction. Recovery of added internal standards was found to be always >90% during a preliminary internal calibration; no corrections were therefore made for extraction efficiency in subsequent analyses. The energy charge ratio, EC, was calculated as ([ATP]+[ADP])/([ATP]+[ADP]+[AMP]) according to Atkinson (1968).
Root respiration
Root respiration rates were determined on excised roots as O2 consumption rates during incubation in 33 ml incubation flasks. Five to ten apical roots with fine laterals (length approximately 40 mm) were excised from each replicate (n=5) and placed in the incubation flasks in freshly prepared nutrient solution at the growth pH. Five to eight glass beads were added to each flask to secure stirring during the incubation, and the flasks were then closed gas-tight and mounted on a rotating wheel in a thermostated (20 °C) water bath. After 1 h, the incubations were stopped, and the oxygen concentration in the flasks analysed using the Winkler method (Montgomery et al., 1964) followed by potentiometric titration with 0.02 N Na2S2O3 to –320 mV. Blinds containing no roots were always included. The roots were dried to constant weight at 80 °C and weighed (0.02–0.04 g dry weight). Weight-specific rates of respiration were calculated from initial and final oxygen concentrations.
Estimating uptake kinetics
The NH4+ and NO3– uptake kinetics were estimated using a modified Michaelis-Menten model (Barber, 1979; Brix et al., 1994):
where In is the inflow rate at substrate concentration Cs, Cs is the substrate concentration in the root medium, Vmax is the maximum influx rate at saturating substrate concentration, Cmin is the substrate concentration in solution at which there is no net inflow, i.e. In=0, and K equals Cs–Cmin where In=Vmax. The half saturation constant, K, is thus equal to K+Cmin. The kinetic parameters Cmin, K and Vmax were estimated by fitting the experimental data during depletion to the model, using a computerized non-linear parameter estimation procedure employing the Gauss-Newton method (Bard, 1967; Brix et al., 1994; Dyhr-Jensen and Brix, 1996). The model described the NH4+ and NO3– depletion satisfactorily in all experiments.
Statistics
Multifactor analysis of variance (MANOVA) using Type III sum of squares was performed with the software Statgraphics version 4.1 (Manugistics, Inc., MD, USA) on the data in order to provide a statistical comparison between the treatment means. Heterogeneities of variances within treatments were tested using Cochran’s C-test. Where necessary, data were transformed to reduce within-treatment heterogeneity. Multiple comparisons of means were performed using the Tukey Honestly Significant Differences (HSD)-procedure at the 5% significance level.
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Plant growth and mineral composition
At the final harvest the size of the plants ranged between less than 50 g fresh weight for the low pH treatments to more than 200 g fresh weight in the other treatments, but the relative distribution between shoots (
35%), roots (5%) and rhizomes (60%) remained almost constant during the experiments, and was not affected by growth-pH and N-source (data not shown). The relative growth rates (RGR) were also constant within treatments during the experiment, but both N-source (P <0.001) and pH (P <0.001) affected RGR significantly, and the effects of pH differed with N-source as indicated by the highly significant interaction term in the ANOVA (P <0.001). For NH4+-fed plants the RGR was highest at pH 6.5 and decreased with lower and higher pH, whereas for NO3–-fed plants the pattern was less clear (Table 1). At pH 3.5 the RGRs were low, only about one-tenth of the highest growth rates obtained at pH 6.5 and 5.0 for NH4+ and NO3–, respectively. At pH 6.5 and 7.0 the RGRs of NH4+-fed plants were significantly higher than the growth rates of NO3–-fed plants, whereas at lower pH no difference between N-source occurred.
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The concentration of total N in the plant tissues generally remained at levels considered adequate for optimal growth (Marschner, 1995), the only exception being rhizomes of NH4+-fed plants at pH 3.5 (850 µmol g–1 dry weight) and shoots of NO3–-fed plants at pH 3.5 (780 µmol g–1 dry weight). The nitrogen concentration in the plant tissue of NH4+-fed plants was generally higher than that of NO3–-fed plants (Fig. 1; Table 2). The nitrogen concentrations increased with growth-pH for NH4+-fed plants, whereas for NO3–-fed plants the nitrogen concentration was highest at pH 5.0 in all plant fractions and lower at other pH treatments. Besides tissue-N, the N-source significantly affected the tissue concentrations of P, Ca, Fe, S, Na, and B, which all occurred in higher concentrations in the NH4+-fed plants (Fig. 1; Table 2). The growth-pH significantly affected the tissue concentrations of Ca, Mg, S, and Na, which were lower in the low-pH treatments, and that of Fe, Cu and Mo, which were higher in the low-pH treatments. The effects were, however, not always identical for NH4+ and NO3–-fed plants, as indicated by the significant interactions between N-source and growth-pH in the ANOVA (Table 2). As an example, the tissue P and S concentrations increased with pH for the NH4+-fed plants, but were highest at pH 5.0 for NO3–-fed plants. The concentration of several of the analysed elements differed between the plant fractions. The concentrations of Ca, Mg and Na were highest in the shoots, whereas the concentrations of P, Fe, Mn, S, Si, Cu, Zn, and Mo were highest in the roots (data not shown).
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Adenine nucleotides
Both N-source and growth-pH significantly affected the content of adenine nucleotides in the plants. Leaves of NH4+-fed plants tended to have (not always statistically significant) higher concentrations of ATP, AMP and TAN than leaves of NO3–-fed plants, but in the roots the pattern was less clear (Table 3). Roots of NH4+-fed plants grown at pH 7.0 had low contents of adenine nucleotides as had also roots of plants grown at pH 3.5. Roots of NO3–-fed plants, however, maintained relatively high levels of adenine nucleotides at pH 7.0, but levels were very low at pH 3.5, and much lower than for NH4+-fed plants. The effects of growth-pH and N-source on the contents of adenine nucleotides in the plant tissues were not alike in the treatments as shown by the significant interaction terms in the ANOVAs (Table 4). The ATP/ADP ratios were highly variable within treatments, but were higher in roots of NH4+-fed plants than in roots of NO3–-fed plants. The energy-charge ratio (EC ratio) in leaves increased with growth-pH irrespective of N-source, as did the EC ratio in roots of NH4+-fed plants. No clear pattern was observed for roots of NO3–-fed plants.
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Ammonium and nitrate uptake kinetics
Both ammonium and nitrate uptake kinetics of T. latifolia were affected significantly by growth-pH (Fig. 2; Table 5). The overall mean values (±95% CL) of the NH4+ uptake kinetic parameters were: Vmax: 22.6±3.6 µmol h–1 g–1 root dry weight; K
: 2.9±1.1 µmol l–1, and Cmin: 0.8±0.3 µmol l–1. The overall mean values (±95% CL) of the NO3– uptake kinetic parameters were: Vmax: 20.3±5.6 µmol h–1 g–1 root dry weight; K: 9.0±3.7 µmol l–1, and Cmin: 2.3±0.5 µmol l–1. Thus, the nitrogen uptake capacity as estimated by Vmax did not differ significantly between NH4+ and NO3–-fed plants, but the affinity for nitrogen as estimated by K and Cmin was significantly lower for NO3–-fed plants than for NH4+-fed plants. The uptake capacity (Vmax) for NH4+ was highest (30.9 µmol h–1 g–1 dry weight) for plants grown at pH 6.5, and lowest (14.0 µmol h–1 g–1 dry weight) for plants grown at pH 3.5 (Fig. 2). For NO3– the uptake capacity was highest (31.7 µmol h–1 g–1 dry weight) for plants grown at pH 5.0, and lowest (4.7 µmol h–1 g–1 dry weight) for plants grown at pH 3.5 (Fig. 2). The affinity for NH4+ was highest at pH 7.0 (K= 1.3 µmol l–1; Cmin=0.5 µmol l–1), and for NO3– at pH 5.0 (K= 1.7 µmol l–1; Cmin=1.2 µmol l–1). Thus, the optimum pH for NH4+ uptake was close to neutral whereas the optimum pH for NO3– uptake was around pH 5.0 (Fig. 2).
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The maximum uptake capacity (Vmax) was significantly correlated to the relative growth rate of both NH4+ (r2=0.95, P=0.03) and NO3–-fed (r2=0.96, P=0.03) plants (Fig. 3A), indicating a linear relationship between growth rate and nitrogen uptake capacity. Root respiration was significantly affected by both N-source (F-ratio=28.7, P <0.001) and growth-pH (F-ratio=30.6, P <0.001), and the effects were not significantly different in the treatments as indicated by the non-significant interaction term in the ANOVA (F=2.3, P=0.09). The respiration rates were generally higher in NH4+-fed plants than in NO3–-fed plants (Table 6), and respiration rates were very depressed at low pH, particularly for NO3–-fed plants. Root respiration rates of both NH4+-fed and NO3–-fed plants were positively related to maximum uptake capacities (Vmax, Fig. 3B) and the relative growth rates (Fig. 3C). The data for NO3–-fed plants grown at pH 6.5 were considered as outliers and thus excluded from the regression analyses in Fig. 3B, C.
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Proton balance
The H+-extrusion during growth with NH4+ was found to differ only between pH 3.5 and the other pH treatments (Table 6). At pH 3.5, there was a net uptake of 1.65 H+ per NH4+ ion taken up as shown by the negative ratio, whereas the average net extrusion at pH 5.0, 6.5, and 7.0 was 1.55 H+ per NH4+ ion taken up. During growth with NO3– plants at all pH treatments had a net uptake of protons (or a corresponding net extrusion of hydroxyl ions). Again, there were no significant differences between pH 5.0, 6.5 and 7.0 where the average net uptake of H+ was 0.35 H+ per NO3– ion taken up. At pH 3.5 the rate of NO3– uptake was low, but the net uptake of protons remained high making the ratio between net proton uptake and nitrate uptake very high (Table 6).
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Discussion |
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The long-term growth response of T. latifolia to N-supply shows that this species grows better with NH4+ as compared with NO3– as the sole N-source. At near-neutral pH-levels NH4+-fed plants had higher relative growth rates, higher tissue concentrations of the major nutrients, higher contents of adenine nucleotides, and a higher affinity for uptake of inorganic nitrogen than NO3–-fed plants. Typha latifolia was, however, able to grow with NO3– as the sole N-source, and grew best at slightly acid pH-levels. At very acid conditions (pH 3.5) the growth rate was very low, the tissue concentrations of the major nutrients and adenine nucleotides were severely reduced, and the uptake capacity for inorganic nitrogen was low, and more so for NO3–-fed plants than for NH4+-fed plants. These effects, and the observed relationship between relative growth rate and nitrogen uptake capacity, support the hypothesis that the inhibition of growth of plants at low pH is due to a decline in nutrient uptake and a consequential limitation of growth by nutrient stress (Tolley-Henry and Raper, 1986; Vessey et al., 1990).
The effects observed at low pH were probably caused directly by the high H+-activity in the root solution, since there appeared to be a large net influx of H+ into the root cells at pH 3.5 for both NH4+ and NO3–-fed plants. The influx of H+ could be caused by an increased permeability of the plasma membrane or an inadequate H+-ATPase pumping activity at low pH, disrupting electrochemical membrane potential and the internal pH regulation of the root cells (Yan et al., 1992). The H+ extruded or taken up during uptake of NH4+ and NO3–, respectively, are produced during the assimilation into organic N compounds, and related to intracellular charge and pH regulation by the plants (Raven and Smith, 1976). Several studies have thus shown an acidification of the root medium during uptake of NH4+, whereas uptake of NO3– generally results in an alkalinization (Marschner and Römheld, 1983; Sorrell and Orr, 1993). The ratio of H+ extruded to NH4+ taken up was slightly above the theoretically calculated value of 1.1–1.25 by Raven and Smith (1976) for pH values above 3.5. The ratio between H+-uptake and NO3– was lower than the expected range of 2 H+ per NO3– taken up (Mistrik and Ullrich, 1996) assuming a 2:1 co-transport mechanism for NO3–. However, the ratio depends on whether the assimilation of NO3– occurs mainly in the shoots or roots (Raven and Smith, 1976; Mistrik and Ullrich, 1996).
The effects of pH on the uptake kinetics of NH4+ and the ratio between NH4+ uptake and net H+ extrusion seen in this study are consistent with the hypothesis that a high H+ activity in the external solution makes conditions more unfavourable for the extrusion of H+ ions that drive the NH4+ uptake. At low pH the efficiency of the H+-efflux pump decreases and, therefore, contribute to the decrease in NH4+ uptake (Yan et al., 1992). A decreased membrane potential at lower pH will, however, facilitate the uptake of NO3– (Marschner, 1995), which is also seen in the present study at pH levels down to pH 5. The reasons for the differences in uptake kinetic parameters for NH4+ and NO3– uptake with root medium pH are still not fully known. The changes may result from changes in the nature and/or relative abundances of the various transporters at the plasma membrane, due to the action of endogenous regulatory mechanisms, that ultimately are controlled by the external conditions of the plant.
The large influx of H+ at pH 3.5 could possibly be due to a limitation in energy supply to the membrane-bound electrogenic proton pumps as the concentrations of adenine nucleotides in the roots were depressed. The energy charge ratio of plant material describes, to some extent, the physiological state of the tissue, and a ratio between 0.8 and 0.9 reflects physiologically vigorous and healthy tissue (Atkinson, 1968). As the tissue is subjected to increasing levels of stress, the energy charge ratio normally decreases and a ratio below 0.5 is usually associated with lethal stresses. The energy charge ratios of the roots were fairly variable in the present study and did not differ statistically significantly between pH treatments. At pH 3.5, however, the ratio was lower for roots of NH4+-fed plants than for NO3–-fed plants supporting the view that NH4+ nutrition is more stressful than NO3– nutrition at very acid conditions.
Unfortunately, experiments in solution cultures (or agar) do not always reflect the actual processes occurring in soils, because buffering and transport reactions in soils differ markedly from those in the nutrient solutions. Typha latifolia is thus found growing in wetlands receiving acid mine drainage with pH levels at 3.4–3.5 (Wieder et al., 1990), where, as judged from the present study, growth would be severely inhibited. Many wetland plants growing in soil are, however, able to create large gradients in oxygen concentration, pH and nutrient concentrations around the roots in the rhizosphere (Marschner and Römheld, 1983; Sorrell and Orr, 1993). In aluminium-tolerant species a plant induced pH increase of the rhizosphere, due to changes in the ratio between cation and anion uptake, is often seen compared with less aluminium-tolerant species (Taylor and Foy, 1985). A plant-induced increase in pH of the rhizosphere could, therefore, be a likely mechanism for increasing the tolerance of T. latifolia to growth at low pH. In addition, an oxygenation of the rhizosphere, as seen in many wetland species (Sorrell et al., 1993; Jespersen et al., 1998; Chabbi et al., 2000), could increase the tolerance towards low pH. In an oxygenated rhizosphere, nitrification of NH4+ to NO3– can occur, and as uptake of NO3– is driven by a cotransport of H+, the pH in the apoplast and the rhizosphere may increase (Sorrell and Orr, 1993) as a result of the net-uptake of H+ from the surroundings or, which is more likely, from a changed internal ion composition because of the requirement for electrical neutrality in the cytosol (Gerendas and Schurr, 1999). Some wetland plants may, therefore, be able to avoid exposure to extreme acid and high NH4+ conditions, at least around the root tips, which are the most sensitive to proton toxity (Koyama et al., 2001). The development of iron-rich coatings (plaques) on the surface of roots (Taylor et al., 1984) may also help prevent exposure to extreme pH levels.
In conclusion, T. latifolia seems to be well adapted to growth in wetland soils where ammonium is the prevailing nitrogen compound, but very low pH levels around the roots are very stressful for the plant. The common occurrence of T. latifolia in very acidic areas is probably only possible because of the plant’s ability to modify pH conditions in the rhizosphere.
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Acknowledgement |
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This study was supported by the Danish Natural Science Research Council.
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