Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez

Efeitos de diferentes fontes amoniacais sobre o metabolismo de nitrogênio da bromélia atmosférica Tillandsia pohliana Mez

Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez

VÍVIAN TAMAKI¹ and HELENICE MERCIER¹,²

(received: January 11, 2001; accepted: July 19, 2001)

ABSTRACT - (Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez). Increasing levels of atmospheric ammonia from anthropogenic sources have become a serious problem for natural vegetation. Short-term effects of different ammoniacal sources on the N metabolism of Tillandsia pohliana, an atmospheric bromeliad, were investigated. One-year-old, aseptically grown plants were transferred to a modified Knudson medium lacking N for three weeks. Plants were subsequently transferred to Knudson media supplemented with 0.5, 1.0, or 1.5 mM of N in the forms of NH₃ or NH₄⁺ as the sole N source. The activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH-NADH) were determined after 40 h. The GS activity was stimulated significantly by increasing the levels of the gaseous form. The GDH-NADH activity increased significantly under increasing N concentrations with NH₃, while no significant differences were observed with NH₄⁺ as a N source. These results may reflect a faster NH₃ absorption by T. pohliana compared to NH₄⁺ uptake. The increased activity of GDH-NADH in NH₃ treatment may play a role in protecting the cells from the toxic effects of increased endogenous level of free ammonium. A raise in the concentration of N, especially in the form of NH₃,greatly increased the content of free amino acids and soluble proteins. A possible utilisation of T. pohliana to evaluate the changes of atmospheric gaseous ammonia is proposed.

RESUMO - (Efeitos de diferentes fontes amoniacais sobre o metabolismo de nitrogênio da bromélia atmosférica Tillandsia pohliana Mez). As fontes antropogênicas de amônia atmosférica têm se tornado um problema sério para a vegetação natural. No presente trabalho foram analisados os efeitos da aplicação de diferentes fontes amoniacais sobre o metabolismo do nitrogênio de Tillandsia pohliana, uma bromélia atmosférica. As sementes foram germinadas in vitro e as plantas cresceram em meio de cultura de Knudson por um ano. Após esse período, elas foram transferidas para o meio de Knudson sem as fontes nitrogenadas originais. Três semanas depois, as plantas foram colocadas nesse mesmo meio de cultura, porém, suplementado com 0,5, 1,0 ou 1,5 mM de N nas formas de NH₃ ou NH₄⁺ como fontes únicas de nitrogênio. O tempo de exposição a essas fontes foi de 40 h. As atividades enzimáticas da glutamina sintetase (GS) e da glutamato desidrogenase (GDH-NADH) foram analisadas. Os teores endógenos de amônio, de aminoácidos livres e de proteínas solúveis, presentes nos eixos caulinares dessa bromélia, também, foram determinados. As atividades da GS e da GDH-NADH foram estimuladas, significativamente, com o aumento do nitrogênio na forma gasosa. Esses resultados podem refletir a rápida absorção de NH₃ pela planta. Entretanto, a atividade aminante da GDH não apresentou diferença significativa em resposta ao aumento do N na forma de NH₄⁺. Provavelmente, o incremento na atividade da GDH-NADH, no tratamento com NH₃, está relacionado com a proteção das células aos possíveis efeitos tóxicos e altas concentrações endógenas de NH₄⁺. O aumento na concentração do nitrogênio, especialmente na forma de NH₃, elevou os conteúdos de aminoácidos livres e de proteínas solúveis. Uma possível aplicação prática para o presente trabalho é a utilização de T. pohliana como indicadora da variação da concentração de amônia atmosférica.

Key words - Bromeliaceae, NH₃, NH₄⁺, GS, GDH-NADH

Introduction

Nitrogen (N) can be released from the soil in the form of ammonia gas (NH₃). NH₃ is the most important alkaline component of the atmosphere (Hanstein & Felle 1999) and most NH₃ emission occurs in areas of intense agricultural activities, where large quantities of fertilisers are employed (Schjørring 1998). It is estimated that about 60% of the global NH₃ emission comes from anthropogenic sources. Emitted NH₃ returns to the surface mainly in the form of dry deposition of NH₃ and wet deposition of NH₄⁺ (Asman et al. 1998). It is possible that throughfall is rich in NH₄⁺ as it was observed in forests in the polluted regions of The Netherlands, with concentrations of 0.32 to 2.4 mM (Van Breemen et al. 1982). In several European countries, it was observed that 50% of the N compounds released into the environment consisted of NH₃/NH₄⁺, and this indicates the importance of these compounds for vegetation (Pearson & Stewart 1993). Besides being released from the soil, NH₃ can be emitted from senescing plant parts (Hanstein & Felle 1999). In addition, plants give off N-NH₃, through the stomata, as has been observed in rice (Oryza sativa L.) (Silva & Stutte 1981), wheat (Triticum aestivum L.) (Parton et al. 1988), and pea (Pisum sativum L.) (Betelsen & Jensen 1992), as well as in several wild species, such as Mercurialis perennis L., Rubus fruticosus L., and Trientalis europaea L. (Pearson et al. 1998). There is strong evidence that increased N level in the atmosphere is beneficial to epiphytic plants up to a certain limit (Baxter et al. 1992). Levels above a limit can poison epiphytes; this particularly applies to lower plants such as lichens and mosses (Baxter et al. 1992).

GS is a central biological catalyst in N metabolism. It catalyses the reaction of NH₄⁺ with glutamate to glutamine with the aid of ATP (Choi & Kwon 1998). GDH-NADH also operates in the assimilation of NH₄⁺ thereby complementing the enzymes of the GS-GOGAT cycle in the synthesis of glutamate, especially under conditions of stress (Srivastava & Singh 1987).

Tillandsia pohliana (figure 1), which belongs to Bromeliaceae, is an atmospheric epiphytic species, which possesses succulent leaves totally covered with epidermic trichomes (figures 2 and 3). The roots operate as holdfast devices, and nutrition occurs through the foliar trichomes, which absorb both water and nutrients (Benzing et al. 1976, Nyman et al. 1987). The importance of trichomes is also associated with light reflection, which, when raised, act as efficient reflectors, softening the thermic action of sun rays (Benzing et al. 1978).

In this study we present a comparison of ammoniacal N utilisation (NH₃ or NH₄⁺) by shoots of T. pohliana cultivated in vitro. The activities of the GS and GDH-NADH enzymes as well as the endogenous concentrations of free NH₄⁺, free amino acids and total soluble proteins were verified. The results obtained have allowed us to build up a picture of the nitrogen metabolism occurring in this atmospheric bromeliad in the context of increasing atmospheric N.

Material and methods

In vitro plant cultivation - Seeds of the atmospheric epiphytic bromeliad Tillandsia pohliana Mez were aseptically germinated in vitro. The medium used contained Knudson's basic salts (1946) and was supplemented with Murashige & Skoog's micronutrients (1962), 2% gelrite, and 3% sucrose. The culture flasks were kept on shelves illuminated with fluorescent daylight lamps (40 mmol.m^-2.s^-1) under a 12 h photoperiod. The growth room temperature was 26 ± 2 °C. In vitro germination and aseptic bromeliad culture were necessary to avoid contamination by nitrifying organisms and the conversion of ammonium to nitrate.

Utilization of different forms of ammoniacal nutrition - One-year-old plants of T. pohliana were transferred to a modified Knudson medium lacking N. Three weeks later, one group of plants was transferred to a modified Knudson medium supplemented with (NH₄)₂SO₂ as the only N source. Three concentrations of this salt were used in order to obtain 0.5, 1.0 and 1.5 mM of N.

Another group of plants was transferred to flasks containing modified Knudson media lacking N. NH₃ was injected into these flasks (figure 4) through a rubber lid which avoids leakage and prevents the exchange of gases with the outside air. The same three N concentrations (0.5, 1.0, and 1.5 mM N) of that gas were used. The ratio of NH₃ dissolution and protonation in the medium surface is dependent on two factors: gelling agent concentration and exposure time. NH₄⁺ formation was considerably decreased when raising the gelrite concentration from 2 to 10% (data not shown). Above 10% the medium was very solid and plant inoculation was difficult. Therefore, 10% of gelrite was used in the experiments where NH₃ was the only N source. Figure 5 shows NH₃ concentration changes after its injection into the flasks, which contained only gellified medium without any plants. NH₃ uptake by the medium with consequent NH₄⁺ formation was less than 10% 40 hours after its injection into the flasks. No NH₃ was detected in the flasks atmosphere after two weeks. Based on these preliminary experiments, 40-hour exposure period was chosen as being sufficient time for foliar absorption with minimal decrease in NH₃ concentration in the flasks.

Shoots of T. pohliana of both experimental groups were collected after a 40-hour exposure to NH₃ gas (the fumigation was initiated during the dark period when the stomata were open) or (NH₄)₂ SO₄ salt and then frozen in liquid N₂ and subsequently stored at -20 °C.

Free ammonium analysis - Extracts were obtained from 1 g of shoots homogenized with 5 ml of water and centrifuged at 11,000 g for 15 min. The supernatant was collected and used for the free-NH₄⁺ analysis.

Free- NH₄⁺ was determined by the phenol-hypochlorite reaction in which 1 ml of the extract was used (Weatherburn 1967). The reaction used a 5 ml of reagent A (containing 10 g phenol and 50 mg sodium nitroprusside in 1 liter of H₂O) and 5 ml of reagent B (containing 5 g NaOH and 8.4 ml NaOCl 5-6%). The reaction tubes were incubated at 25 °C for 30 min and the absorbance was read at 625 nm. The NH₄⁺ concentration was expressed as nmol NH₄⁺ g^-1 FM.

The process of NH₄⁺ determination described above (the phenol-hypochlorite reaction) was used for the analysis of NH₄⁺ in the gellified medium (20 ml of the medium were used).

Enzyme extraction - One gram of shoots was ground in liquid N₂ and centrifuged (at 12,000 g for 40 min at 4 °C) with 5 ml of a buffer solution (0.05 M imidazole and 5 mM DTT, pH 7.9). The supernatant was centrifuged at 12,000 g for 40 min at 4 °C. After that, the supernatant was maintained at 4 °C and used for all the enzymatic assays (Cammaerts & Jacob 1985).

Glutamine synthetase (GS) assay - GS (EC 6.3.1.2) activity was measured as described by Pérez-Soba et al. (1994) and Farnden & Robertson (1980) with modifications for T. pohliana.

The reaction mixture with a final volume of 500 ml contained 1.2 M hydroxylamine, 0.32 M glutamate, 40 mM MgCl₂, 50 mM ATP (pH 7.5) and 150 ml of the plant extract. The tubes were incubated at 30 °C for 40 min. The final step of the reaction was the addition of 1 ml of a reagent containing 0.37 M FeCl₃, 0.20 M trichloroacetic acid, and 0.67 M HCl. The absorbance of the samples was measured at 540 nm. Blank controls were analysed with a 0.5 ml imidazole buffer (0.1 M, pH 7.5) and 1 ml of the reagent described above. The GS activity was expressed as mmol g-GHA.min^-1 g^-1. FM.

Glutamate dehydrogenase - aminating (GDH-NADH) assay - GDH-NADH (EC 1.4.1.2) activity was determined spectrophotometrically at 340 nm as described by Cammaerts & Jacobs (1985), modified for T. pohliana. The activity was determined according to NADH oxidation.

The assay mixture consisted of 0.15 M (NH₄)₂ SO₄, 1 mM CaCl₂, 0.14 mM NADH, 0.02 M 2-oxoglutarate, 0.5 ml 0.1 M Tris-HCl pH 8.2, and 0.5 ml of plant extract. The Sephadex G-25 column was essayed, but the enzyme activity did not show any difference. The reaction was incubated at 30 °C for 10 min. Blank controls without the plant extract and 0.02 M 2-oxoglutarate were used. The GDH-NADH activity was expressed as mmol NADH. min^-1. g^-1 FM.

Total free amino acids analysis - One gram of shoots was homogenised with 20 ml of 80% ethanol. The homogenate was passed through four layers of gauze and one layer of Whatman N^o 1 filter paper. Pigments and proteins were extracted by partition with chloroform and water as described by Fernandes (1983), and the total free amino acids were assayed by the ninhydrin method (Moore & Stein 1954). The data were expressed as mmol leucine. g^-1 FM.

Protein analysis - The proteins were extracted as previously described and determined according to the procedure proposed by Bradford (1976). The levels of soluble proteins were expressed as mg albumin. g^-1 FM.

Statistical analysis - Data were subjected to analysis of variance. The Tukey test was used to compare the means. Significant differences were inferred when P < 0.05.

Results and Discussion

The results of the endogenous levels of free NH₄⁺ in the shoots showed a faster absorption of N in the form of NH₃ in comparison with NH₄⁺ (figure 6). In concentrations higher than 0.5 mM, T. pohliana absorbed NH₃ faster than NH₄⁺. T. pohliana's shoots were fumigated with NH₃ during the dark period when its stomata were open, since it is a CAM bromeliad (Tamaki & Mercier 1997). This procedure favored a net influx of NH₃ into the leaves, which caused rapid changes in N metabolism. This bromeliad does not seem to be susceptible to damage after 40 h of exposure to NH₃. The use of another epiphytic bromeliad, Tillandsia usneoides, as an air pollution indicator has shown that this species is an efficient accumulator of atmospheric mercury (Malm et al. 1998). It was highlighted by these authors that since T. usneoides is a CAM bromeliad, its tolerance to drought stress makes it more appropriate than bryophytes and lichens for biomonitoring tropical environments.

Plant leaves can take up not only nitrate and ammonium, but also atmospheric gaseous NH₃ and utilise it as a source of N (Farquhar et al. 1980, Schjørring et al. 1993). NH₃ and NH₄⁺ can enter plants through the stomatal openings, by micropores that exist on the cuticule that are readily permeable to solutes (Marschner 1995), and by the special foliar trichomes that exist in bromeliads (see figure 2 and 3). NH₃ is highly reactive and forms soluble products inside the leaves as can be exemplified by the following equation: NH₃ + H₂O « NH₄⁺ + OH^-. The major determinants for NH₃ deposition are stomatal conductance and environmental conditions, particularly humidity and light that increase the rate of deposition (Van Hove et al. 1987).

When NH₃ enters the leaves it gets dissolved in the apoplasmic solution, and NH₄⁺ formation is a very rapid process. Its transport into the cytoplasm of leaf cells may occur by carrier-mediated exchanges and an increase in NH₄⁺ concentration in the leaves would be expected after fumigation with NH₃ (Yin et al. 1996). That increase in NH₄⁺ concentration was confirmed in T. pohliana shoots (figure 6). The build-up of free NH₄⁺ in the plant tissues may not necessarily inhibit photosynthesis or growth on a short-term basis. The acid-base regulation in cells depends on intrinsic features of the plant's metabolism (Pearson & Stewart 1993).

The results obtained for T. pohliana clearly demonstrated that the GS enzyme was stimulated by increasing the concentrations of N in the gaseous form (NH₃). At the concentration of 1.5 mM, the gas induced the highest GS activity (figure 7A). The aminating activity of the GDH enzyme did not show any variation as a result of the increase in the N concentration in the form of NH₄⁺ (figure 7B). However the GDH-NADH activity increased with a rise in NH₃, which suggests that the aminating function of this enzyme is restricted to the use of this form of N. The increased activity of GDH-NADH may play a role in protecting the cells from the toxic effects of increased levels of free NH₄⁺. Such increase in GDH activity as a function of an increase in N level has also been reported in Arabidopsis thaliana by Cammaerts & Jacobs (1985) and in Vitis vinifera by Loulakakis & Roubelakis-Angelakis (1992). On the other hand, GDH was not found to be modulated by N concentration in rice (Magalhães & Huber 1991) and Kalanchöe lateriria (Santos & Salema 1992). Perez-Soba et al. (1994) observed that exposure of Pinus sylvestris L. shoots to NH₄⁺ caused increases in the concentrations of free amino acids and soluble proteins, but did not affect GDH-NADH activity.

Levels of amino acids and soluble proteins in shoots of T. pohliana increased regardless of the N form used (figures 8A and B). The gaseous ammoniacal source was the most inductive. The influence of different N compounds on the production of amino acids in plants has been widely studied. In the case of various plant materials it has been shown that a co-relationship exists between the increase of N availability and the production of free amino acids and proteins (Fernandes 1983, Santos & Salema 1991, Perez-Soba et al. 1994, and Leport et al. 1996). The amino acid, which is often present in greater proportions, as a result of an increase in N concentration, is glutamine, as reported by Barneix et al. (1984) in barley (Hordeum vulgare L. cv. Sonja), and by Sugiharto & Sugiyama (1992) in maize (Zea mays L.).

A possible practical application of the present work could be the use of T. pohliana plants as ammoniacal pollution indicators. Their morphology and physiology (as observed in the N-metabolism characterisation presented in this study) make them suitable for handling since they are totally independent from the soil, and for transplanting to different areas where air quality measurements are necessary.

Ackowledgments - The first author thanks the CNPq for a graduate-level scholarship, and we thank MSc Cristiane Kalife for the schematic view of an individual trichome.

2. Autor para correspondência: [e-mail]

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1

. Universidade de São Paulo, Instituto de Biociências, Laboratório de Fisiologia Vegetal, Caixa Postal 11461, 05422-970 São Paulo, SP, Brasil.

Publication Dates

• Publication in this collection
  31 July 2002
• Date of issue
  Dec 2001

History

• Accepted
  19 July 2001
• Received
  11 Jan 2001