Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 51, No. 343, pp. 227-237, February 2000
© 2000 Oxford University Press

Rapid effects of nitrogen form on leaf morphogenesis in tobacco

Pia Walch-Liu^1,4, Günter Neumann¹, Fritz Bangerth² and Christof Engels³

¹ Institut für Pflanzenernährung, Universität Hohenheim, D-70593 Stuttgart, Germany
² Institut für Obst-, Gemüse- und Weinbau, Universität Hohenheim, D-70593 Stuttgart, Germany
³ Abteilung Agrarökologie, Fachgruppe Geowissenschaften der Universität Bayreuth, D-95440 Bayreuth, Germany

Received 15 April 1999; Accepted 16 September 1999

	Abstract

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Ammonium (NH⁺₄) instead of nitrate (NO^-₃) as the nitrogen (N) source for tobacco (Nicotiana tabacum L.) cultivated in a pH-buffered nutrient solution resulted in decreased shoot and root biomass. Reduction of shoot fresh weight was mainly related to inhibition of leaf growth, which was already detectable after short-term NH⁺₄ treatments of 24 h, and even at a moderate concentration level of 2 mM. Microscopic analysis of the epidermis of fully expanded leaves revealed a decrease in cell number (50%) and in cell size (30%) indicating that both cell division and cell elongation were affected by NH⁺₄ application. Changes in various physiological parameters known to be associated with NH⁺₄-induced growth depression were examined both in long-term and short-term experiments: the concentrations of total N, soluble sugars and starch as well as the osmotic potential, the apparent hydraulic conductivity and the rate of water uptake were not reduced by NH⁺₄ treatments (duration 1–12 d), suggesting that leaf growth was neither limited by the availability of N and carbohydrates, nor by a lack of osmotica or water supply. Although the concentration of K⁺ in leaf press sap declined in expanding leaves by approximately 15% in response to NH⁺₄ nutrition, limitation of mineral nutrients seems to be unlikely in view of the fast response of leaf growth at 24 h after the start of the NH⁺₄ treatment. No inhibitory effects were observed when NH⁺₄ and NO^-₃ were applied simultaneously (each 1 mM) resulting in a NO^-₃/NH⁺₄ net uptake ratio of 6 : 4. These findings suggest that the rapid inhibition of leaf growth was not primarily related to NH⁺₄ toxicity, but to the lack of NO^-₃-supply. Growth inhibition of plants fed solely with NH⁺₄ was associated with a 60% reduction of the zeatine+zeatine riboside (Z+ZR) cytokinin fraction in the xylem sap after 24 h. Furthermore Z+ZR levels declined to almost zero within the next 4 d after start of the NH⁺₄ treatment. In contrast, the concentrations of the putative Z+ZR precursors isopentenyl-adenine and isopentenyl-adenosine (i-Ade+i-Ado) were not affected by NH⁺₄ application. Since cytokinins are involved in the regulation of both cell division and cell elongation, it seems likely that the presence of NO^-₃ is required to maintain biosynthesis and/or root to shoot transfer of cytokinins at a level that is sufficient to mediate normal leaf morphogenesis.

Key words: Ammonium, nitrate, leaf growth, cytokinin.

	Introduction

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Nitrogen (N) is a major limiting nutrient for plants in many ecosystems. It is taken up from soils mainly as nitrate (NO^-₃) and/or ammonium (NH⁺₄) by the roots of higher plants. Although N assimilation is associated with reduction of NO^-₃ to NH⁺₄, many plants show growth inhibition when NH⁺₄ is supplied as the exclusive N source (Gerendás et al., 1997; Raab and Terry, 1994; Magalhäes and Huber, 1989; Jungk, 1977; Kirkby and Hughes, 1970). Growth inhibition has been attributed to various factors, such as NH⁺₄-induced disorders in pH regulation and toxic effects of free ammonia (Claussen and Lenz, 1995; Goyal et al., 1982; Givan, 1979; Puritch and Barker, 1967; Barker et al., 1966), deficiency of mineral nutrients, such as K⁺ (Barker et al., 1967), Ca²⁺ (Wilcox et al., 1973) and Mg²⁺ (Jungk, 1977), and to carbohydrate limitation (Cramer and Lewis, 1993; Breteler, 1973) due to excessive consumption of soluble sugars for NH⁺₄ assimilation (detoxification). The lack of NO^-₃ as an important osmoticum (Raab and Terry, 1995; Salsac et al., 1987; Chaillou et al., 1986) and water deficit in response to a reduction in water uptake (Adler et al., 1996; Quebedeaux and Ozbun, 1973) have also been reported as putative growth-limiting factors in plants supplied with NH⁺₄ as the sole N source. Moreover, there is some evidence that the N form may affect the endogenous levels of phytohormones such as cytokinins (Wang and Below, 1996; Gao et al., 1992; Smiciklas and Below, 1992), known to be implicated in the regulation of morphogenesis (Kende and Zeevaart, 1997).

In the present work, long-term and short-term effects of N form on leaf growth, on the status of carbohydrates, water and mineral nutrients, as well as on speciation and translocation of cytokinins in tobacco grown in a pH-buffered nutrient solution with moderate NH⁺₄ supply have been investigated. As far as is known, this is the first report on rapid responses of plant growth induced by the application of different forms of N below the level of NH⁺₄ toxicity.

	Materials and methods

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Plant cultivation
Tobacco (Nicotiana tabacum L. cv. Samsun and cv. Gatersleben) was grown in a climate chamber under controlled environmental conditions with a 14 h light period (light intensity of 450 µE m^-2 s^-1), a 25/20 °C light/dark temperature regime, and 60% relative humidity. Tobacco seeds were germinated in a mixture consisting of 90% (w/w) peat culture substrate (Euflor GmbH, München, Germany), 7% (w/w) perlite and 3% (w/w) sand. About 2 weeks after sowing, plants were transferred to an aerated, hydroponic culture system, and supplied with saturated CaSO₄ solution during the first 24 h, and thereafter with a full-strength nutrient solution consisting of H₃BO₃ 10 µM, MnSO₄ 0.5 µM, ZnSO₄ 0.5 µM, CuSO₄ 0.1 µM, (NH₄)₆Mo₇O₂₄ 0.01 µM, Fe-EDTA 15 µM, KH₂PO₄ 0.5 mM, MgSO₄ 1.2 mM, CaCl₂ 2.0 mM. Nitrogen was applied either as KNO₃ or (NH₄)₂SO₄ at a concentration of 2 mM N or as mentioned in the text. For NH⁺₄ or NH₄NO₃ treatments, K₂SO₄ was added to compensate for potassium applied in the KNO₃ variants. Complete N depletion in the nutrient solution was avoided by checking the N concentration in the solution at least once a day using a RQflex reflectometer (Merck, Darmstadt, Germany) and by adding appropriate N amounts. The nutrient solution was renewed completely every 2 d and pH was held between 6.8 and 7.2 by addition of CaCO₃.

Leaf area and expansion
Leaf length and width were determined daily at the same time using a ruler and leaf area (A) was calculated assuming an ellipsoid leaf shape using the formula:

where a is the leaf lengthx0.5, b is the leaf widthx0.5. Leaf expansion rate was expressed as the increase in leaf area h^-1.

Cell size and cell number
Size of epidermal cells was estimated by microscopic analysis of cellulose acetate or nail varnish imprints. Leaf discs of about 1 cm diameter were pressed on to acetone-soaked cellulose acetate sheets supported on a glass slide. After several minutes, when the cellulose acetate sheet had dried, the leaf discs were gently removed. Leaf squares of about 2.5x4 cm were pressed on to a nail varnish-soaked glass slide (Hampe, 1979). Photographs were taken from distinct areas and the average cell size was calculated based on cell counts per unit leaf area. Cell number per leaf was determined by extrapolation of cell number per unit leaf area to the total leaf area.

Carbohydrate analysis
Reducing sugars and sucrose were determined according to a modified method of Blakeney and Mutton (Blakeney and Mutton, 1980). About 20 mg of freeze-dried plant material was extracted in 5 ml of 70% (v/v) ethanol by swirling in a tube and centrifuged at 2500 g (Minifuge, Hereaus GmbH, Stuttgart, Germany). Chlorophyll in leaf extracts was removed by addition of activated charcoal (about 10 mg ml^-1), swirling and centrifuging at 18 000 g (Mikro Centrifuge, Hettich, Tuttlingen, Germany). For determination of reducing sugars, 0.2 ml of the clear supernatant was mixed with 0.8 ml 0.1 M sodium acetate (pH 4.8). Sucrose was determined by mixing 0.1 ml of supernatant with 0.1 ml invertase solution (50 units ml^-1 buffered in 0.2 M sodium acetate) and 0.8 ml 0.1 M sodium acetate (pH 4.8). The mixture was incubated for 2 h in a 30 °C water bath to digest sucrose to glucose and fructose. 5 ml of colour reagent (0.03 M hydroxybenzoic acid hydrazide, 0.05 M tri-sodium citrate, 0.01 M calcium di-chloride, and 0.5 M sodium hydroxide) was added to the sample solutions (sucrose and reducing sugars) and boiled for 4 min in a water bath. The cooled coloured solution was measured spectrophotometrically at 415 nm (AA spectrometer U-3300, Hitachi, Tokyo, Japan). Starch was determined in the residual pellet (according to Blakeney and Matheson, 1984). Starch was dissolved in 2 ml di-methyl sulphoxide by boiling for 10 min in a water bath. The sample was centrifuged at 2500 g (Minifuge, Hereaus GmbH, Stuttgart, Germany) and the pellet was washed with 8 ml 0.1 mM sodium acetate (pH 4.8) solution. 1 ml of the sample solution was mixed with 2 ml amyloglucosidase solution (1.2 U ml^-1 buffered in 0.2 M sodium acetate) and incubated for 12 h at 37 °C in a water bath. After incubation, 5 ml of colour reagent (4000 U glucose oxidase, 1000 U peroxidase, 0.07 M di-sodium hydrogen phosphate, 0.04 M sodium di-hydrogen phosphate, 0.016 M benzoic acid, 0.5 mM 4-amino antipyrin, 0.01 M p-hydroxybenzoic acid) was added to each 1 ml sample solution and kept in a water bath at 40 °C for 15 min. The cooled coloured solution was measured spectrophotometrically at 510 nm (AA spectrometer U-3300, Hitachi, Tokyo, Japan).

Nitrogen analysis
Total N was estimated in freeze-dried plant material with a NCS 2500 Elemental Analyser (CE Instruments, Milan, Italy) using Dumas combustion. The sample is energetically oxidized yielding a gas mixture in which N is detected by a thermoconductivity detector. Nitrate was determined in a water extract of the sample (according to Britt, 1962) using a Technicon II Auto Analyser (Technicon, Dublin, Ireland)). The freeze-dried plant material (10 mg) was mixed with deionized water (10 ml) and filtered through Blue Ribbon filter paper (Schleicher & Schuell GmbH, Dassel, Germany). Nitrogen net uptake was measured by applying ¹⁵N (5% enrichment) in the nutrient solution as K¹⁵NO₃, NH₄¹⁵NO₃ or ¹⁵NH₄NO₃ for 4 h and determining the ¹⁵N enrichment in the plant tissue over time. ¹⁵N/¹⁴N isotopes were analysed by coupling the Dumas principle with a stable isotope mass spectrometer (Roboprep-CN and Tracermass, Europa Scientific Ltd, Crewe, UK). Total N net uptake was calculated by multiplying the amount of ¹⁵N taken up with a correction factor for the % ¹⁵N supplied and divided by time and root fresh weight.

Press sap analysis
Press sap of young, expanding and fully expanded leaves was analysed for K⁺ and Ca²⁺ by flame emission photometry (ELEX 6361, Eppendorf, Hamburg, Germany), Mg²⁺ by atomic absorption spectrometry (AAspectrometer Unicam 939, ATI UNICAM, Kassel, Germany), Cl^- using a chloridemeter (chloridemeter 6610, Eppendorf, Hamburg, Germany) and NO^-₃ using the auto analyser procedure as described above, or by using a RQflex reflectometer (Merck, Darmstadt, Germany). The osmotic potential of leaf press sap was determined using an osmometer (automatisches Halbmikro-Osmometer Typ Digital, Knauer, Berlin, Germany).

Collection of xylem exudate and determination of apparent hydraulic conductivity
For collection of xylem exudate and the determination of water flux, plant shoots were cut 2 cm above the root/shoot interface. After at least 15 min, the cut stem was cleaned with paper tissue to avoid contamination with contents of wounded cells and phloem exudate. Thereafter, xylem exudate was collected with a Pasteur pipette for approximately 1 h (storage on ice) and subsequently stored at -20 °C. Water flux was calculated from the quotient of collected volume of xylem exudate and the collecting time. The osmotic potentials of xylem exudate and nutrient solution were determined using an osmometer.

The apparent hydraulic conductivity was calculated after the formula of Fiscus (Fiscus, 1975):

where Lp is the apparent hydraulic conductivity, J_v is the water flux (ml h^-1), is the reflection coefficient, P_x is the pressure of xylem exudate (MPa), and P_a is the pressure of external solution (MPa). It was assumed that was not affected by the form of N supply, and for the calculation of Lp was set=1.

Determination of water uptake
Water uptake was determined gravimetrically after a preculture with NH⁺₄ or NH₄NO₃ (2 mM N) followed by 2 d of NH⁺₄ or NO^-₃ treatments (2 mM N), respectively. Plants were removed from the culture vessels (1 plant per pot), and the weight of the pots was recorded after complete draining of nutrient solution from the root systems. The daily rate of water uptake was calculated from the differences in weight compared to the start of the experiment. Background evaporation was negligible since the pots were kept completely closed with lids throughout the entire experimental period.

Analysis of cytokinins
Xylem exudate was collected as described above and stored at -80 °C. Cytokinins (CKs) were determined (according to Bangerth, 1994). The exudate was purified by adjusting the pH to 8.5 and passing it first over a polyvinylpyrrolidone column and then, after adjusting the pH to 3.0, over a Waters C-18 Sep Pak cartridge (Waters, Milford, Mass. USA). The cartridge was then washed with 0.1 M acetic acid and zeatin (Z) and zeatinriboside (ZR) were eluted with 4 ml of 25% (v/v) methanol in 0.1 M acetic acid; finally isopentenyladenine+isopentenyl-adenosine (i-Ade+i-Ado) were eluted with 70% (v/v) methanol. After vacuum evaporation, the purified CKs were quantified by a radioimmunoassay (as described by Bohner and Bangerth, 1988). All immunoassays were performed in triplicate.

Statistical analysis
The statistical software Sigma Stat Version 2.03 (SPSS Inc.) was used for analysis of variance. Comparisons among the means were conducted using Student–Newman–Keuls test.

	Results

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N form effects on growth
Tobacco plants examined in this study did not exhibit any visible symptoms of NH⁺₄ toxicity such as marginal necrosis and interveinal chlorosis on the leaves, wilting, stunted root growth or brownish roots (Goyal et al., 1982; Maynard and Barker, 1969; Barker et al., 1966) (Figs 1, 2).

View larger version (69K): [in this window] [in a new window]   Fig. 1. Effect of N form on shoot growth of tobacco (cv. Samsun) cultivated in nutrient solution at 2.5 mM N supplied with NH+4 or NO-3 for 11 d before harvest (46 DAS).

 

View larger version (68K): [in this window] [in a new window]   Fig. 2. Effect of N form on the shoot growth of tobacco (cv. Samsun) cultivated in nutrient solution at 2.5 mM N supplied with NH4NO3 or NO-3 for 11 d before harvest (46 DAS).

 
The fresh weights of shoot and roots in two cultivars of tobacco (cv. Samsun and cv. Gatersleben) were significantly reduced when NH⁺₄ instead of NO^-₃ was supplied as the N source (Table 1; Fig. 1). Shoot biomass production was mainly affected by inhibition of leaf growth, whereas the number of unfolding and unfolded leaves was only slightly decreased (Table 1). Microscopic analysis of the epidermis of fully expanded leaves revealed a 50% reduction of cell number and a 30% reduction of cell size in NH⁺₄-treated plants (Table 2; Fig. 3).

View this table: [in this window] [in a new window]   Table 1. Growth characteristics of tobacco as affected by the N form: (a) cv. Samsun supplied with 2.5 mM N for 11 d before harvest (DAS 46), (b) cv. Gatersleben supplied with 1.5 mM N for 10 d before harvest (DAS 32) Unfolded and unfolding leaves were counted starting from the oldest leaf (first). Leaf area of the youngest expanded leaf (10th) is shown. Data are mean values ±se of four replicate plants. Differences between N treatments were statistically tested by ANOVA and means in the same column with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

View this table: [in this window] [in a new window]   Table 2. Effect of N form on leaf area (cm2), epidermal cell size (mm2) and cell number per leaf of the youngest fully expanded leaf of tobacco (cv. Samsun) cultivated in nutrient solution at 2 mM N supplied with NO-3 or NH+4 for 12 d before harvest (40 DAS) Data are mean values ±se of four replicate plants. Differences between N treatments were statistically tested by ANOVA and means in the same column with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

View larger version (168K): [in this window] [in a new window]   Fig. 3. Effect of N form on epidermis cells (cellulose acetate imprints) of fully expanded leaves of tobacco (cv. Samsun) cultivated in buffered nutrient solution at 2.5 mM N supplied with NO-3 (A) or NH+4 (B) for 11 d before harvest (42 DAS). The scale-bar is 100 µm.

 
Growth inhibition exhibited a rapid response to NH⁺₄ application. Transferring the plants to a nutrient solution with NH⁺₄ as the sole N source, after a preculture with NO^-₃–N, resulted in a significant reduction of leaf growth within 24 h. Similarly, stimulation of leaf growth was detectable 24 h after reapplication of NO^-₃–N to plants formerly grown with a sole NH⁺₄ supply (Table 3).

View this table: [in this window] [in a new window]   Table 3. Short-term effect (24 h) of N form on leaf expansion rate (cm2 h-1) after transfer of tobacco plants from NO-3 containing nutrient solution to either NO-3 or NH+4 supply at 2 mM N: (a) cv. Samsun (39 DAS) and (b) cv. Gatersleben (27 DAS) Leaves were counted starting with the oldest (first). Data are mean values ±se with four (cv. Samsun) or five (cv. Gatersleben) replicate plants. Differences between N treatment within each group (genotype and leaf number) were tested by ANOVA and means in the same column within each group (genotype and leaf number) with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 
No inhibition of leaf growth was detectable when NH⁺₄ and NO^-₃ were applied simultaneously at concentration levels of 2.5 mM (Table 1; Fig. 2). ¹⁵N-uptake studies revealed similar net uptake rates of total N for plants supplied with NO^-₃ or NH₄NO₃ (Fig. 4). Mixed N supply resulted in a 6 : 4 net uptake ratio for NO^-₃ and NH⁺₄ (Fig. 4). Obviously, there was no complete discrimination of NH⁺₄ net uptake by the presence of NO^-₃ at the supplied 1 : 1 ratio.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effect of N form on net uptake rates of NO-3 (open columns) and NH+4 (hatched columns) of tobacco (cv. Gatersleben). Nitrogen was supplied at 2 mM either as K15NO3 or NH415NO3 or 15NH4NO3. Columns represent mean uptake rates of plants 3 and 5 d after transfer from preculture with NH4NO3 (28 and 30 DAS) to solutions containing either NO-3 or NH4NO3. Vertical bars represent the standard error of the mean (n=10).

 

N form effects on plant concentrations of carbohydrates and mineral nutrients
To assess putative effects of NH⁺₄ toxicity at the physiological level, the responses of various physiological parameters known to be affected by NH⁺₄ toxicity were recorded in NH⁺₄-treated tobacco plants. Concentrations of N and soluble sugars, which are the main substrates for leaf growth and expansion, were not reduced by supply of NH⁺₄ instead of NO^-₃ as the sole N source (Table 4). Starch and sugar concentrations in shoot tissue even increased in response to NH⁺₄ supply (Table 4), suggesting that shoot growth was not affected by N and C substrate limitation. Concentrations of K⁺, Mg²⁺ and Ca²⁺ in leaf press sap of old leaves decreased by approximately 30–50% in response to NH⁺₄ application (Table 5). In the press sap of young (about 15% of full size) and expanding leaves (about 40% of full size) K⁺ concentration decreased only by about 15%, while Mg²⁺ and Ca²⁺ concentrations did not change or even slightly increased (Table 5). The plants did not show any visible symptoms of mineral nutrient deficiency.

View this table: [in this window] [in a new window]   Table 4. Effect of N form on the concentrations of N, sugars (glucose, fructose, sucrose) and starch in shoot and roots of tobacco (cv. Samsun). Data are means (±se, n=8) of plants cultivated at 0.5 mM or 5 mM N supplied as NO-3 or NH+4 for 12 d before harvest (42 DAS) The N concentrations of the nutrient solutions had no effect on the concentrations of N and carbohydrates in the plants. Therefore mean values of both N supply concentrations are given in the table. Differences between NO-3 and NH+4 nutrition were tested statistically by ANOVA and means in the same column with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

View this table: [in this window] [in a new window]   Table 5. Effect of N form on the osmotic potential and ion concentrations in the press sap of old (fully expanded), expanding (about 40% of full size) and young leaves (about 15% of full size) of tobacco (cv. Samsun) cultivated in nutrient solution at 2.5 mM N supplied as NO-3 or NH+4 or both for 14 d before harvest (53 DAS) Data are mean values ±se (n=8). Differences between the N forms within each group (leaf stage) were tested statistically by ANOVA and means in the same column within each group (leaf stage) with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

N form effects on the water status
The osmotic potential in press sap of young and expanding leaves was not changed by sole NH⁺₄ supply (Table 5). Nitrate, an important osmoticum, was detectable only in plants supplied with NO^-₃. Ammonium application resulted in a 2-fold increase in the leaf concentration of Cl^-, whereas the K⁺ concentration, quantitatively the most important cation, decreased by approximately 20%. Water supply of the plants, as estimated by the apparent hydraulic conductivity of the roots, was not affected by the form of N supply, neither 1 d nor 5 d after transfer from NH₄NO₃ to NO^-₃ or NH^-₄ nutrition (Fig. 5). In the present experiment apparent hydraulic conductivity of the roots was calculated with the assumption that membrane permeability for solutes (reflection coefficient ) was not affected by the N form. Supply of different N forms over 2 d also had no effect on the water uptake (Fig. 6), which was not even changed when NH⁺₄ was applied as sole the N source during the preculture period (Fig. 6).

View larger version (38K): [in this window] [in a new window]   Fig. 5. Effect of N form on the apparent hydraulic conductivity of tobacco roots (cv. Gatersleben) 1, 3 or 5 d after transfer of plants from NH4NO3 to solutions containing either NO-3 (open columns) or NH+4 (hatched columns) supply before harvest (27, 29 or 31 DAS). Data are mean values and vertical bars represent standard error of the mean (n=5). Differences between N form at each harvest time were tested by ANOVA. Means with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

View larger version (37K): [in this window] [in a new window]   Fig. 6. Effect of the N form on water uptake of tobacco (cv. Samsun) 2 d after transfer from NH+4 or NH4NO3 during preculture to either NO-3 (open columns) or NH+4 (hatched columns) supply at 2 mM N (46 DAS). Data are mean values and vertical bars represent standard errors (n=4). Differences between N forms were tested by ANOVA. Means with different letters are significantly different (P<0.05, Student-Newman-Keuls test).

 

Short-term effect on xylem exudate composition
After preculture with NH₄NO₃, the NO^-₃ concentration in xylem exudates decreased rapidly about 40-fold within 24 h when NH⁺₄ was applied as sole N source. This was associated also with a drop in NO^-₃ concentrations in leaf and root tissue (Table 6). Similarly, NH⁺₄ application decreased the zeatin and zeatin riboside (Z+ZR) fraction of cytokinins in xylem exudates within 24 h by 70%. Five days after starting the NH⁺₄ treatment, the Z+ZR levels had declined to almost zero (Table 7). In contrast, the concentrations of isopentenyl-adenine and isopentenyl-adenosine (i-Ade+i-Ado), which are putative precursors of Z+ZR, remained constant or even slightly increased in response to NH⁴₊ application (Table 7).

View this table: [in this window] [in a new window]   Table 6. Short-term effect (24 h) of N form on NO-3 concentration in xylem exudate and NO-3 content of plant tissue of tobacco (cv. Gatersleben) after transfer of tobacco plants from NH4NO3 to either NO-3 or NH+4 supply at 2 mM N (27 DAS) Data are mean values ±se (n=5). Differences between N form were tested by ANOVA and means in the same column with different letters are significantly different (P<0.05, Student–Newman–Keuls test).

 

View this table: [in this window] [in a new window]   Table 7. Effect of the supplied N form after 1, 3 or 5 d on the concentration of the active form of cytokinins, zeatin+zeatin riboside (Z+ZR) and isopentenyladenine+isopentenyladenosine (i-Ade+i-Ado), in xylem exudate of tobacco (cv. Gatersleben) after transfer of plants from NH4NO3 to either NO-3 or NH+4 supply at 2 mM N (27 DAS) Data are mean values ±se of five replicate plants. Differences between N form were tested by ANOVA and means in the same column within each group (cytokinin form) with different letters are significantly (P<0.05, Student–Newman–Keuls test).

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
Effects of NH⁺₄ supply on leaf expansion
Ammonium induced inhibition of shoot growth has been reported for various plant species such as Phaseolus vulgaris L., Beta vulgaris L., Lycopersicon esculentum Mill., and Zea maize L. (Raab and Terry, 1994; Chaillou et al., 1986; Cramer and Lewis, 1993; Magalhäes and Huber, 1989). However, there are also contradictory reports for some species (Oryza sativa L., Calluna vulgaris L., Potentilla erecta L.), which were found to be tolerant to sole NH⁺₄ nutrition (Troelstra et al., 1995; Magalhäes and Huber, 1989).

In this study with tobacco, inhibition of shoot growth in response to NH⁴₊ application could be attributed mainly to a reduction in leaf area and not to a decreased number of leaves (Table 1). Similar results have been reported for Beta vulgaris L. (Raab and Terry, 1994). Reduced leaf area may be due to reduced cell number (MacAdam et al., 1989) and/or smaller cell size (Snir and Neumann, 1997; Palmer et al., 1996; Taylor et al., 1993). Leaf growth of NH⁺₄-treated tobacco was found to be limited by both reduced cell number and cell expansion (Table 2). Most remarkably, growth inhibition was already detectable 24 h after the start of the NH⁺+₄ treatment, and was quickly reversible within 24 h of reapplication of NO^-₃.

Growth depression due to NH⁺₄ toxicity?
Growth reduction in response to application of NH⁺₄ as the sole N source has been frequently related to NH⁺₄ toxicity associated with acidification, uncoupling of photophosphorylation, lack of carbohydrates or mineral nutrients and impairment of the water status. Excess uptake of cations as a consequence of NH⁺₄ nutrition is balanced by an increased net efflux of protons. The resulting acidification of the root environment can lead to inhibition of root growth and even to a destruction of the root tissue (Claussen and Lenz, 1995; Goyal et al., 1982; Maynard and Barker, 1969). To prevent over-acidification of the growth medium in our experiments, the nutrient solution was continuously buffered around pH 7.0 by addition of CaCO₃. At neutral or alkaline pH, however, the NH₃/NH⁺₄ ratio in the nutrient solution increases. In contrast to NH⁺₄ ions, plasmalemma permeation of ammonia (NH₃) is mediated by diffusion, which cannot be controlled by the plant. As a consequence, accumulation of toxic intracellular NH₃ levels has been reported in response to NH⁺₄ nutrition (Tillberg et al., 1977; Avron, 1960), which was related to disturbances in the regulation of the intracellular pH, uncoupling of photophosphorylation and a reduction of photosynthesis (Raab and Terry, 1994). However, recent studies have shown that NH⁺₄ supply at moderate concentrations (3 mM) had no negative effects on intracellular pH regulation (Bligny et al., 1997). Increased tissue concentrations of starch and soluble sugars reported in the present study (Table 4) also suggest that photosynthesis was not directly affected by NH⁺₄ application. However, in the long-term, photosynthesis might be decreased by a feedback repression in response to increased sugar accumulation, which has been similarly reported for N deficiency (Paul and Driscoll, 1997).

It has been frequently stated that root growth and, therefore, whole plant growth of NH⁺₄-fed plants is restricted by low availability of carbohydrates due to excessive consumption of soluble sugars for NH⁺₄ assimilation (detoxification) in the root tissue (Cramer and Lewis, 1993; Kafkafi, 1990; Breteler, 1973). However, in the present work with tobacco (similar to the reports of Kandlbinder et al., 1997; Chaillou et al., 1986), NH⁺₄ application increased the accumulation of soluble sugars both in shoot and root tissue. Furthermore, there was no effect on total N concentration in shoot and roots (Table 4). Therefore the present results suggest that growth of NH⁺₄-fed tobacco was not limited by C and N availability. In the long-term, expanding leaves of NH⁺₄-fed plants showed a 15% decrease in K⁺ concentrations, but no decrease in Mg²⁺ and Ca²⁺ concentrations (Table 5). A decrease in cation concentrations is in accordance with results by other authors (Jungk, 1977; Wilcox et al., 1973; Barker et al., 1967). However, considering the rapid inhibition of leaf expansion within 24 h after starting the NH⁺₄ treatment (Table 3), it is unlikely that growth was primarily restricted by NH⁺₄-induced deficiency of mineral nutrients in the present experiment.

There are various reports postulating that growth reduction of NH⁺₄-fed plants might be caused by a lack of NO^-₃ as an important osmotic anion for leaf cell expansion (Raab and Terry, 1994; Salsac et al., 1987; Chaillou et al., 1986). In the present study, however, the osmotic potential of leaf press sap was not changed in response to NH⁺₄ application (Table 5). The absence of NO^-₃ was obviously compensated for by increased accumulation of chloride, which was also a quantitatively important osmotic compound even in NO^-₃-fed tobacco plants (Table 5). Similar results have been reported for barley supplied with NH⁺₄ as N source (Soltani et al., 1989). The analysis of bulk leaf press sap reflects overall N form-dependent differences in the osmotic potential of the leaf tissue. However, the technique cannot account for spatial or subcellular variations of ion concentrations in different tissues and cell compartments (Fricke et al., 1994; Miller and Smith, 1996). Therefore, growth depression due to the lack of osmotica cannot be entirely excluded. Several authors suggested that reduced hydraulic conductivity of the roots in response to NH⁺₄ application may contribute to water limitation, which affects leaf expansion by turgor reduction (Adler et al., 1996; Quebedeaux and Ozbun, 1973). Estimations of the apparent root hydraulic conductivity (Fig. 5) and water uptake (Fig. 6) in tobacco plants did not reveal significant changes due to application of different N forms. Similarly, it was found that water uptake was not affected by NO^-₃ or NH⁺₄ supply (Goyal et al., 1982). Therefore, NH⁺₄-induced inhibition of leaf growth as a result of limitation in water is not supported by the present data.

In summary, it may be concluded that, in this study, the rapid and reversible inhibitory effects on leaf growth in tobacco supplied with moderate NH⁺₄ concentrations were not mediated by NH⁺₄ toxicity.

Cytokinin levels
The most striking effect of sole NH⁺₄ supply to tobacco plants was a rapid decrease in the zeatin+zeatin riboside (Z+ZR) cytokinin fraction in xylem exudates (Table 7). It is well known that xylem exudate data do not reflect translocation in intact transpiring plants (Else et al., 1995). However, as water uptake rates in intact plants were not affected by the form of N supply (Fig. 6), it can be assumed that the decrease in xylem sap Z+ZR concentration reduced Z+ZR supply from the roots to the shoot. In accordance with this assumption shoot tissue Z+ZR concentration in NH⁺₄-fed plants dropped to approximately 30% compared with NO^-₃ containing nutrient solution (data not shown). Both phenomena were associated with the rapid inhibition of leaf growth. In xylem sap, a 70% decrease of Z+ZR was already detectable 24 h after starting the NH⁺₄ treatment, and decreased to almost zero within the next 4 d. These results are in contrast to other authors (Bubán et al., 1978) who showed that cytokinins in the xylem exudate of apple rootstocks were more enhanced by NH⁺₄ than by NO^-₃ supply. There are, however, obvious similarities to the rapid decline in cytokinin levels in xylem sap and leaf tissue, associated with inhibition of leaf expansion in response to N deprivation (Wagner and Michael, 1971; Sattelmacher and Marschner, 1978; Palmer et al., 1996). Nitrogen has been suggested to be an effector of cytokinin production (Samuelson et al., 1992; Parkash, 1982; Sattelmacher and Marschner, 1978), for which root meristems are an important source (Letham and Palni, 1983). Cytokinin supply to the shoot is mainly mediated via xylem transport, although root-independent cytokinin production in above-ground vegetative tissue has been demonstrated at least for transgenic plants (Faiss et al., 1997). Constant, or even slightly increased levels of the putative Z+ZR precursors, isopentenyl-adenine+isopentenyl-adenosine (i-Ade+i-Ado) (Chen, 1997), in the xylem sap of NH⁺₄-treated tobacco plants suggest that the reduction in Z+ZR levels may be either attributed to an inhibition of i-Ade+i-Ado conversion to Z+ZR or to selective inhibition of Z+ZR loading into the xylem.

Several authors have discussed the importance of cytokinins in regulating biomass partitioning between shoot and root (Fetene and Beck, 1993; Kuiper et al., 1988). Fetene and Beck found that exogenous cytokinin supply to the roots stimulated shoot growth by increasing carbon partitioning towards the shoot (Fetene and Beck, 1993). There is also evidence that exogenously supplied cytokinins promote leaf expansion independent of uptake and utilization of exogenously supplied carbohydrates (Nielsen and Ulvskov, 1992). Cytokinins can promote both cell division (Taiz and Zeiger, 1998) and cell expansion (Rayle et al., 1982). Passage of cells through phases of the mitotic cycle is controlled by a family of auxin-induced serine/threonine protein kinases and their regulatory subunits, the cyclins (Kende and Zeevaart, 1997). There is evidence that cytokinin increases the abundance of cyclin mRNA (Soni et al., 1995). Recently, it was reported that the product of a cytokinin-inducible soybean mRNA (Cim1) is located to the cell wall with homology to a subfamily of expansin proteins (Downes and Crowell, 1998). Expansins are probably involved in cell expansion as catalysts, mediating loosening of cell walls (Cosgrove, 1998). There is also evidence for both synergistic and antagonistic interactions of cytokinins with auxins, which are involved in the regulation of cell expansion (Coenen and Lomax, 1997). Therefore, it seems likely that growth inhibition of tobacco leaves in response to NH⁺₄ nutrition (Table 3), which was associated with a reduction in both cell number and cell size, is mediated by decreased root-to-shoot translocation of cytokinins (Z+ZR).

The involvement of plant growth regulators, such as cytokinins, in leaf morphogenesis is further supported by the rapid induction of the N-form responses. The striking similarities to the effects of N limitation suggest that growth inhibition by NH⁺₄ nutrition is related rather to the absence of NO^-₃ than to the presence of NH⁺₄. Thus, apart from its function as N source, NO^-₃ may be involved in a signal transduction chain that regulates leaf morphogenesis by supplying the shoot with cytokinins. Both root and leaf NO^-₃ levels rapidly decreased to almost zero within 24 h in response to NH⁺₄ application. However, it is still an open question as to whether the primary response to the absence of NO^-₃ is located in root or leaf tissue. Moreover, it is also possible that other hormonal factors such as abscisic acid, which has been reported to increase in xylem sap, leaf and root tissue of NH⁺₄-treated Ricinus communis plants (Peuke et al., 1994), mutually interact with cytokinins in the regulation of leaf morphogenesis.

	Acknowledgments

 
This paper is dedicated to the memory of Professor Dr Dres hc Horst Marschner. We would like to thank Gisela Moll (Institut für Phytomedizin, Universität Hohenheim, Germany) for her friendly help with the microscopic photography and Heidi-Jayne Hawkins and Dr Sam Alvey for critical reading of the manuscript. This project was financially supported by DFG (Deutsche Forschungsgemeinschaft).

	Notes

 
⁴ To whom correspondence should be addressed. Fax: +49 711 459 3295. E-mail: piawalch{at}uni\|[hyphen]\|hohenheim.de

	Abbreviations

 
CK, cytokinin; DAS, days after sowing; i-; Ade, isopentenyl-adenine; i-Ado, isopentenyl-adenosine; Z, zeatine; ZR, zeatine riboside..

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