Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

Aquatic Botany 92 (2010) 142–148

Contents lists available at ScienceDirect

Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot

Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates Dennis Konnerup *, Hans Brix Department of Biological Sciences, Plant Biology, Aarhus University, Ole Worms Alle´ 1, DK-8000 Aarhus C, Denmark

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 May 2009 Received in revised form 29 October 2009 Accepted 11 November 2009

The effects of inorganic nitrogen (N) source (NH4+, NO3 or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05–0.06 g g1 d1), biomass allocation and plant morphology of C. indica were indifferent to N nutrition. However, NH4+ fed plants had higher concentrations of N in the tissues, lower concentrations of mineral cations and higher contents of chlorophylls in the leaves compared to NO3 fed plants suggesting a slight advantage of NH4+ nutrition. The NO3 fed plants had lower light-saturated rates of photosynthesis (22.5 mmol m2 s1) than NH4+ and NH4+/NO3 fed plants (24.4–25.6 mmol m2 s1) when expressed per unit leaf area, but similar rates when expressed on a chlorophyll basis. Maximum uptake rates (Vmax) of NO3 did not differ between treatments (24–35 mmol N g1 root DW h1), but Vmax for NH4+ was highest in NH4+ fed plants (81 mmol N g1 root DW h1), intermediate in the NH4NO3 fed plants (52 mmol N g1 root DW h1), and lowest in the NO3 fed plants (28 mmol N g1 root DW h1). Nitrate reductase activity (NRA) was highest in leaves and was induced by NO3 in the culture solutions corresponding to the pattern seen in fast growing terrestrial species. Plants fed with only NO3 had high NRA (22 and 8 mmol NO2 g1 DW h1 in leaves and roots, respectively) whereas NRA in NH4+ fed plants was close to zero. Plants supplied with both forms of N had intermediate NRA suggesting that C. indica takes up and assimilate NO3 in the presence of NH4+. Our results show that C. indica is relatively indifferent to inorganic N source, which together with its high growth rate contributes to explain the occurrence of this species in flooded wetland soils as well as on terrestrial soils. Furthermore, it is concluded that C. indica is suitable for use in different types of constructed wetlands. ß 2009 Elsevier B.V. All rights reserved.

Keywords: Constructed wetland Hydroponic Ornamental plant Stomatal conductance Transpiration Vmax Wastewater treatment

1. Introduction Nitrogen (N) nutrition of plants mainly depends on the availability of the different inorganic N forms in the environment, but other factors such as pH and concentrations of mineral ions also affect nutrition (Dyhr-Jensen and Brix, 1996; Romero et al., 1999; Brix et al., 2002; Guo et al., 2007). Terrestrial plants often take up most of their N as nitrate (NO3) as concentrations of ammonium (NH4+) are generally very low in well-drained soils because of the microbial nitrification processes occurring under oxic conditions (Mengel and Kirkby, 2001). However, in soils where the microbial nitrification processes are inhibited, e.g. by low pH or lack of oxygen, NH4+ is the predominating form of inorganic N available for plant uptake (Kronzucker et al., 1997). In waterlogged soils and sediments of aquatic ecosystems, NO3 will

* Corresponding author. Tel.: +45 89424701; fax: +45 89424747. E-mail address: [email protected] (D. Konnerup). 0304-3770/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2009.11.004

usually only occur in high concentrations in the water column and in a thin top layer of the soil (Nijburg and Laanbroek, 1997). Plants growing in wetland soils and waterlogged sediments therefore predominantly take up N in the form of NH4+. For most plants, a mixed NO3 and NH4+ nutrition is superior over sole NO3 or NH4+ nutrition, but the optimal proportions of NO3 to NH4+ depend on plant species, environmental conditions, developmental stage and the concentration of supplied N (Chaillou et al., 1991; Claussen, 2002; Zou et al., 2005; Tylova-Munzarova et al., 2005). The uptake and assimilation of NH4+ has lower energy costs than uptake and assimilation of NO3. However, many plant species show reduced growth under strict NH4+ nutrition and develop NH4+ toxicity symptoms including chlorosis of leaves, overall suppression of growth and lowering of root to shoot ratios (Gerendas et al., 1997). In contrast, a surplus uptake of NO3 can be stored in the vacuoles of the plant cells without harmful effects (Marschner, 1995). The energy requirement for assimilation of the two forms of N differs as NO3 is in the oxidised state and NH4+ is in the reduced

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148

state. Incorporation of N into organic compounds in the plant tissue requires N in the reduced state. Hence, when the plants take up N in the form of NO3, it has to be reduced within the plant cells which is an energy consuming process. First, NO3 is reduced to NO2 by nitrate reductase (NR), and subsequently NO2 is reduced to NH4+ by nitrite reductase. The costs for the reduction of NO3 to NH4+ is lower if the conversion takes place in photosynthetic leaves because it can be coupled directly to the electron transport chain in the light reaction (Raven, 1985). NH4+ fed plants save 7.6–11.9% of the overall cost for growth as compared to NO3 fed plants where NO3 reduction and assimilation occurs in the roots. However, when the NO3 reduction and assimilation occurs in the shoot of the NO3 fed plants, the difference to NH4+ fed plants is reduced to 3.0–6.1% because of the direct use of photons to fuel the NO3 reduction (Gerendas et al., 1997). The overall effects of inorganic N form on plant growth are also dependent on environmental conditions such as irradiance, temperature, N concentrations and medium pH. In some plants, the inhibitory effect of strict NH4+ nutrition on growth is more pronounced under conditions of high irradiance than under low irradiance. At high root temperatures, tolerance of plants to NH4+ is often reduced, and at low pH plants can take up more NO3 since uptake of this ion is a H+/NO3 co-transport whereas uptake of NH4+ is associated with H+ being pumped out of the root cells. Hence, uptake of NO3 is associated with an increase in pH in the surroundings and uptake of NH4+ results in a decrease in pH. Constructed wetlands (CW) are planted beds used for the treatment of various types of wastewater (Brix and Schierup, 1989). There are two main types of CW systems, namely horizontal subsurface flow CWs and vertical flow CWs. In horizontal flow CWs the gravel substrate is water-saturated and N occurs mainly in reduced forms as organic N and NH4+. In vertical flow CWs, the water percolates vertically through unsaturated sand or gravel, and hence oxygen concentrations are high and a substantial fraction of N occurs as NO3 (Kadlec and Wallace, 2008). The plants have several roles in the CWs, including uptake of nutrients which can subsequently be removed through harvest of the aboveground biomass (Brix, 1997). Traditionally, fast-growing wetland species like Phragmites spp. and Typha spp. are used in the CWs. However, recently, and particularly in the tropics, great emphasis has been put on increasing the aesthetics of the systems by using ornamental flowers like Canna spp. and other species (Brix et al., 2007; Konnerup et al., 2009; Zurita et al., 2009). Canna spp. can grow in both terrestrial and aquatic environments but little is known about their nutrient requirements, including their preference and response to difference forms of inorganic N. Hence, the aim of the present study was to assess how Canna indica, which is commonly used in CW systems, responds to NH4+ or NO3 nutrition, and to elucidate N uptake assimilation in the plant. For this purpose, growth, biomass allocation, photosynthesis and nitrate reductase activity in C. indica supplied with NH4+ and NO3 alone or in combination were compared. Furthermore, N uptake rates and mineral concentrations in the different plant fractions were estimated. 2. Materials and methods 2.1. Plant material and growth conditions Rhizome cuttings of Canna indica L. were planted in moist, coarse sand and placed in a growth chamber (Weiss Umwelttechnic, Lindenstruth, Germany) operated at a 12:12 h day:night photoperiod, a 22:20 8C thermo period and 85:90% day:night relative air humidity. The photon flux density at the surface of the substrate was approximately 300 mmol m2 s1 (PAR) provided by metal halide bulbs. After five weeks, 18 new shoots were separated

143

from the rhizomes and transferred to a hydroponic growth system consisting of 4.2-l containers with lids with a central hole for mounting of individual plants. The nutrient solution was prepared from deionised water and reagent grade salts and had the following composition (mM): NH4–N 0.25; NO3–N 0.25; PO4–P 0.1; K 0.18; Ca 0.63; Mg 0.30; Na 0.70; SO4 0.31; Cl 1.2; (103 mM): BO3 0.39; Fe(II) 1.3; Mn(II) 0.76; Zn 0.03; Cu 0.10. The pH of the nutrient solution was adjusted to 7.0 with 1 M NaOH and the solutions were continuously aerated with atmospheric air. A complete solution renewal was undertaken every fourth day in order to minimize nutrient depletion (less than 50%) and changes in pH (less than 0.5 pH-units). After 28 days, the initial fresh weights (FW) of 18 plants were determined and the plants randomly assigned to three experimental treatments (n = 6) in a randomized complete block design with N supplied as NO3, NH4+, or both at equimolar concentrations (0.5 mM). The 0.5 mM NH4+ treatment was prepared from (NH4)2SO4, the combined N treatment from NH4NO3 and the 0.5 mM NO3 treatment from KNO3. Since NO3 was added as KNO3, the two other treatments were supplied with equivalent concentrations of K as K2SO4 in order to obtain equal concentrations of K in all treatments. We did not compensate for the additional SO42 added, as SO42 has been shown not to affect plants significantly at these concentrations (Mengel and Kirkby, 2001). 1 mM CaCO3 was added to the nutrient solutions to buffer against pH changes caused by nutrient uptake. The nutrient solutions were renewed daily, and after 11 days the N and P concentrations in the nutrient solutions were doubled to meet the increased nutrient requirements of the plants. The total duration of the experimental treatment was 23 days. 2.2. Plant growth and biomass allocation Fresh weights of all plants were determined at the beginning and every third day during the experiment using a standardized weighing procedure. Relative growth rates (RGR) were calculated as the difference in the natural logarithm of final and initial fresh weights divided by the growth period in days. At the end of the experiment, all plants were harvested and rinsed in deionised water before they were separated into leaves, stems, rhizomes, roots and new shoots. The fractions were weighted and then dried at 80 8C to constant weight to estimate the FW to dry weight (DW) ratio. 2.3. Photosynthesis, transpiration and stomatal conductance Gas exchange of the second youngest fully expanded leaf was measured after two weeks with an ADC LCA-4 infrared gas analyser (IRGA) equipped with Leaf Microclimate Control System (ADC BioScientific Ltd., UK) at 22 8C. The leaf chamber was supplied with atmospheric air from outside the building at a rate of 400 ml min1 using an air pump. Maximum light-saturated photosynthesis (Pmax) at atmospheric CO2 level was measured with light supplied from a halogen source (Portable Light Unit, PLU-002, ADC BioScientific Ltd., UK) at a photosynthetic photon flux density of 1800 mmol m2 s1. After gas exchange measurements, the leaf was excised, the surface area and FW measured, and then frozen and freeze dried for later pigment analysis. 2.4. N uptake rates Average maximum uptake rates (Vmax) of NH4+ and NO3 were estimated from depletion in the growth solution. At day 22, the nutrient solutions were renewed, and over the following six hours, the depletion in NH4+ and NO3 concentrations were monitored by hourly sampling and analysis of NH4+ and NO3 solution

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148

144

concentrations. The following day, the NH4+ fed plants were placed in vessels with NO3 and no NH4+, and the NO3 fed plants were placed in vessels with NH4+ and no NO3. Uptake of NH4+ and NO3 were then measured again using a similar protocol as before. Concentrations of NH4+ and NO3 in all samples were analysed using a modified salicylate method (Quikchem Method no. 10-10706-3-B, Lachat Instruments, Milwaukee, WI, USA) and the UV method (Oscarson et al., 1988), respectively. Plant uptake rates were calculated by fitting the depletion curve with linear regression analyses. As a lag-phase with lower rates was often observed initially, and as the uptake rate decreased at low solution concentrations, the three points that gave the steepest slope were chosen in each case to estimate Vmax. Root DW were estimated just after the uptake studies, and the uptake rates were expressed on a root DW basis (mmol N g1 root DW h1). 2.5. Nitrate reductase activity At day 19, samples of roots and leaves were harvested from all plants after 6 h in the light and stored in liquid nitrogen before further processing. Nitrate reductase (NR) activity was determined as NO2 accumulation according to the method from Scheible et al. (1997). The extraction buffer used for the analyses contained 100 mM HEPES-KOH (pH 7.5), 5 mM Mgacetate, 1 mM EDTA, 10% (v/v) glycerol, 0.1% Triton X-100, 5 mM DTT, 1% (w/v) BSA and 1% (w/v) PVPP, 20 mM FAD and 5 mM Na2MoO4, 3% (w/v) casein, 25 mM leupeptine, 0.5 mM phenylmethylsulphonyl fluoride and 5 mM o-phenanthrolin. Since it has been shown that the extraction can reduce enzyme activity (Munzarova et al., 2006), the loss of enzyme activity was tested by addition of a commercial NR enzyme from corn seedlings (Sigma) to the extracts, and enzyme recovery rates were calculated. Recovery rates were tested in assays with commercial NR only, with plant extract only, and with commercial enzyme and plant extract combined. Recovery rates were in the range 65–80%. NR activity was analysed as the rate of NO2 production in 50 ml of the assay buffer, pre-warmed to 25 8C and incubated for 2, 5, 10 and 30 min at 25 8C. The linearity of the assay was checked and the optimal duration of the incubation time was set for each plant fraction. The assay buffer contained 100 mM HEPES-KOH (pH 7.5), 5 mM KNO3, 0.25 mM NADH and 5 mM EDTA. The reaction was stopped with 25 ml of 0.6 mM Znacetate and 75 ml 0.15 mM phenazine methosulphate, and samples were kept 15 min at room temperature before the NO2 concentrations were analysed colorimetrically after adding 300 ml of 1% (w/v) sulphanilamide in 3 M HCl and 300 ml of 0.02% (w/v) N-(1-naphthyl)-ethylenediamine in distilled H2O. The colour developed during 20 min at room temperature was measured at 540 nm (Shimadzu Spectrophotometer UV-1700) after centrifugation at 14,000  g (Eppendorf Centrifuge 5417 C, Hamburg, Germany). Enzyme assay mixtures containing Zn-

acetate prior to addition of the plant extracts were used as the time zero controls. 2.6. Plant tissue analysis The concentrations of Chl a and b and total carotenoids in the second youngest leaf which was also used for measurement of photosynthesis, were analysed by extraction of 5–10 mg freeze dried leaves with 96% ethanol and spectrophotometric measurement according to Lichtenthaler (1987). DW and surface area of leaves were determined before analysis, and concentrations of pigments were expressed in mg cm2 leaf. Samples from stems, leaves, rhizomes, roots and new shoots were ground in a ball mill (Mixer Mill MM 400, Retsch, Haan, Germany) and analysed for concentrations of total N and carbon (C) by gas chromatography after combustion of 2–3 mg dried plant material (Fisons Instruments, Model NA2000, Carlo Erba, Italy). The C/N atomic ratio was calculated, and the N use efficiency (NUE) estimated as the total dry plant biomass divided by total N concentration. Leaf NUE was calculated as leaf biomass divided by leaf N concentration. The concentrations of sodium (Na), calcium (Ca), magnesium (Mg), and potassium (K) in the plant tissues were analysed in subsamples (c.150–180 mg) of finely ground freeze dried plant material. The samples were digested in a microwave sample preparation system (Multiwave 3000, Anton Paar GmbH, Austria) using 4 ml concentrated HNO3 and 2 ml H2O2. The concentrations of the elements were analysed by inductively coupled plasmaspectrometry (Optima 2000 DV, PerkinElmer Instruments Inc., CT, USA). 2.7. Statistical analyses Data were analysed by analysis of variance (ANOVA) using Type III sum of squares with the software Statgraphics XV centurion version 15.1.02 (StatPoint, Inc., USA). Multiple comparisons of means were performed using Tukey HSD procedure at the 0.05 significance level. All data were tested for normal distribution by Kolmogorov–Smirnov test and for homogeneity of variance by Bartlett’s test. If necessary, logarithmic or square root transformations were performed to ensure homogeneity of variance, but for clarity all data are presented as untransformed. 3. Results 3.1. Plant growth and biomass allocation The mean growth rates of C. indica in the three treatments were in the range 0.052–0.060 g g1 d1 and were not affected by N form (Table 1). Nitrogen nutrition did also not affect either biomass, biomass allocation or any of the plant morphological parameters measured.

Table 1 Relative growth rates (g g1 d1) and biomass allocation of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days and results of ANOVA (F-ratios). Values are means  S.D. (n = 5–6).

1

1

RGR (g g d ) Biomass (g DW plant1) A/B ratio S/R ratio SLA (cm2 g1 DW) Shoot height (cm) Number of leaves Number of new shoots Number of roots

NH4+

NH4NO3

NO3

F-Ratio

0.057  0.008 22  7 5.1  1.1 7.8  1.2 266  16 53  5 7.6  0.6 4.2  1.5 20  8

0.060  0.006 24  3 3.7  0.3 5.8  0.7 269  11 62  3 8.3  0.5 5.7  0.8 23  7

0.052  0.009 24  7 4.5  2.0 7.1  2.4 259  21 59  8 8.3  0.5 4.3  1.5 23  13

1.5 0.2 1.5 2.1 0.6 3.7 3.4 2.3 0.1

A/B: above ground/below ground; S/R: shoot/root; SLA: specific leaf area; ns: not significant at the 0.05 level.

ns ns ns ns ns ns ns ns ns

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148 Table 2 Tissue nitrogen (N) and carbon (C) concentrations, atomic C/N-ratios and nitrogen use efficiency (NUE) of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days and results of ANOVA (F-ratios). Different letters indicate significant differences between treatments. Values are means  S.D. (n = 5–6). NH4+ Tissue N conc. (% DW) Leaves Stems Rhizomes Roots New shoots

3.8  0.2b 3.1  0.4ab 2.0  0.1b 2.0  0.5 4.9  0.5b

NH4NO3

NO3

145

Table 3 Tissue concentrations of Na, Ca, Mg and K in leaves, stems, rhizomes, roots and new shoots of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days and results of ANOVA (F-ratios). Different letters indicate significant differences between treatments. Values are means  S.D. (n = 5–6).

F-Ratio

4.4  0.3c 3.6  0.5b 1.7  0.3b 2.1  0.3 4.7  0.2b

3.4  0.2a 2.7  0.4a 1.2  0.2a 1.7  0.1 3.8  0.4a

27.5*** 6.7** 15.5*** 2.0 ns 12.5***

Na (mg g1 DW) Leaves Stems Rhizomes Roots New shoots

NH4+

NH4NO3

0.11  0.10 0.57  0.23 3.42  0.95 1.62  0.64 0.13  0.04a

0.19  0.14 0.46  0.06 3.46  1.11 1.20  0.23 0.32  0.16b

0.17  0.08 0.60  0.17 3.31  0.71 1.28  0.32 0.28  0.05ab

0.9 ns 1.3 ns 0.1 ns 1.5 ns 3.9*

NO3

F-Ratio

Tissue C conc. (% DW) Leaves Stems Rhizomes Roots New shoots

43.7  0.4c 37.2  1.0b 43.3  1.3b 39.9  0.7 40.2  0.3b

42.9  0.5b 34.8  2.3ab 41.3  0.7a 39.1  0.6 37.5  0.8a

41.5  0.3a 34.7  0.9a 40.8  0.8a 39.6  0.8 37.1  1.3a

41.9*** 4.2* 9.8** 1.9 ns 18.0***

Ca (mg g1 DW) Leaves Stems Rhizomes Roots New shoots

12.1  1.9a 6.0  0.7a 4.3  0.4 4.5  0.6a 2.2  0.6a

17.0  3.4b 6.7  0.2a 5.0  1.4 4.6  0.4a 3.1  0.4ab

18.7  2.6b 8.7  1.2b 4.8  1.3 6.0  1.3b 4.4  1.5b

9.5** 0.8* 0.5 ns 5.9* 9.3**

C/N-ratio Leaves Stems Rhizomes Roots New shoots

13.5  0.8b 14.4  2.0ab 25.4  2.0a 24.7  5.5 9.7  1.2ab

9.1  4.5a 11.5  1.9a 29.7  5.8a 22.0  2.7 9.3  0.5a

14.5  0.7b 15.7  3.0b 39.2  6.0b 26.8  2.4 11.5  1.6b

34.7*** 5.0* 9.2** 2.6 ns 5.9*

Mg (mg g1 DW) Leaves Stems Rhizomes Roots New shoots

8.2  1.5a 8.1  0.7a 10.4  1.9a 6.8  1.2a 7.6  2.2a

10.1  1.3ab 10.0  0.3b 14.7  3.7b 9.0  1.2b 9.5  1.6a

11.1  1.5b 10.7  0.9b 16.40  2.3b 10.9  1.6b 14.3  1.7b

6.2* 21.9*** 7.9** 14.3*** 18.1***

NUE (g DW g1 N) Whole plant basis Leaf basis

31.2  1.8b 26.5  1.5b

27.4  1.9a 22.6  1.8a

35.8  2.7c 29.9  1.6c

22.2*** 28.9***

54  6 99  10 40  8 71  8 115  19ab

2.6 ns 1.6 ns 2.7 ns 0.0 ns 5.0*

ns: not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

3.2. C/N-ratio, mineral composition and photosynthetic pigments The tissue concentrations of N were affected by N treatment in all plant fractions except roots (Table 2). Plants fed with NH4+ and NH4NO3 generally had higher concentrations of N compared to NO3 fed plants. The C/N-ratio in all fractions, except roots, also differed between treatments, as NO3 fed plants had higher ratios than plants in NH4NO3, and in rhizomes, also higher than in NH4+ fed plants. The N source affected N use efficiency (NUE) significantly (Table 2). NO3 fed plants had the highest NUE and NH4NO3 fed plants the lowest both when NUE was calculated on a whole plant basis and on a leaf basis. The concentrations of Na, Ca, Mg and K were significantly affected by N source and generally NH4+ fed plants had lower concentrations of the four elements (Table 3). The effects were most pronounced for Ca and Mg where NH4+ fed plants had the lowest concentrations in nearly all plant fractions. The contents of Chl a, but not Chl b, in the leaves were affected by N source (Table 4). Plants in the NH4+ and NH4NO3 treatments had mean Chl a concentrations of 9.26 and 9.17 mg g1 DW, respectively, whereas NO3 fed plants had 6.81 mg g1 DW. Since the Chl a differed between treatments, the Chl a/b ratio and total Chl also differed. Total content of carotenoids were also affected by N source with NO3 plants having the lowest concentration (Table 5). 3.3. Photosynthesis, transpiration and stomatal conductance The light-saturated rate of photosynthesis (Pmax) differed between treatments, but intercellular CO2 concentrations (Ci), transpiration rates and stomatal conductances to water vapour (gs) were not affected (Table 6). Plants fed with NO3 had significantly lower Pmax compared to plants fed with NH4+ and NH4NO3. 3.4. N uptake rates Maximum uptake rates (Vmax) of NH4+ were affected by N source (P < 0.001) whereas uptake rates of NO3 did not differ among

K (mg g1 DW) Leaves Stems Rhizomes Roots New shoots

49  7 91  11 33  5 70  8 85  11a

59  8 101  10 43  10 70  9 133  46b

ns: not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

plants from the different treatments (Fig. 1). The uptake rate of NH4+ was highest in the NH4+ fed plants (81.4 mmol N g1 root DW h1), intermediate in the NH4NO3 fed plants (52.4 mmol N g1 root DW h1), and lowest the in NO3 fed plants (28.0 mmol N g1 root DW h1). The average uptake rates of NO3 were in the range 23.8–35.0 mmol N g1 root DW h1 across treatments. 3.5. Nitrate reductase activity Nitrate reductase activity (NRA) was detected in both roots and leaves of plants from all treatments, and the rates differed significantly between roots and leaves and were also affected by N form (Table 6; Fig. 2). The two-way ANOVA showed main effects of N form and plant fraction and it also showed significant interaction between the two main factors. In NH4+ fed plants, NRA was very low in both leaves and roots whereas in the two other treatments,

Table 4 Concentrations of photosynthetic pigments in leaves of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days and results of ANOVA (F-ratios). Different letters indicate significant differences between treatments. Values are means  S.D. (n = 5–6). Photosynthetic pigments

NH4+

NH4NO3

NO3

F-Ratio

Chl a (mg g1 DW) Chl b (mg g1 DW) Chl a/b ratio Total Chl (mg g1 DW) Total Car. (mg g1 DW)

9.26  1.38a 2.74  0.45 3.38  0.20a 12.00  1.81a 2.36  0.37ab

9.17  0.67a 2.59  0.25 3.55  0.14a 11.76  0.90a 2.42  0.15a

6.81  1.28b 2.22  0.49 3.09  0.14b 9.03  1.77b 1.97  0.29b

8.7** 2.59 ns 12.7*** 6.7** 4.3*

ns: not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

146

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148

Table 5 Gas exchange characteristics of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days and results of ANOVA (F-ratios). Different letters indicate significant differences between treatments. Values are means  S.D. (n = 5–6). NH4+

NH4NO3 a

2 1

Pmax (mmol m s ) Intercellular CO2 conc. (mmol mol1) Transpiration rate (mmol m2 s1) Stomatal conductance (mol m2 s1)

24.4  2.1 285  4 8.04  0.46 1.29  0.40

a

25.6  2.3 286  11 8.23  0.58 1.76  1.02

NO3

F-Ratio

22.5  1.7b 291  19 8.03  0.82 1.77  1.44

3.5** 0.3 ns 0.2 ns 0.4 ns

ns: not significant. ** P < 0.01.

there was significantly higher NRA in leaves compared to roots. The highest NRA was observed in leaves of NO3 fed plants with a mean rate of 21.9 mmol NO2 g1 DW h1 which was significantly higher than the mean rate of 7.7 mmol NO2 g1 DW h1 in leaves of plants fed with NH4NO3. 4. Discussion Canna indica grew well with high growth rates independent of the inorganic N source being NH4+ or NO3 supplied alone or in combination. Neither biomass allocation nor any of the morphological parameters measured were affected by the type of inorganic N source, and NH4+ fed plants did not have any NH4+ toxicity symptoms. For comparison, a study of Maranta leuconeura, which is also a tropical species growing in humid and warm conditions, showed a slight preference for NO3 in the young stage, but growth and morphology of older plants were not affected by N source (Strojny, 1999). Two other emergent wetland plants, Phragmites australis and Glyceria maxima showed different growth responses to NH4+ and NO3 nutrition. The growth rate of P. australis was not affected by N source whereas G. maxima had 16% higher growth rate when fed with NH4+ compared to NO3 fed plants (TylovaMunzarova et al., 2005). NH4+ fed plants had higher tissue N concentrations indicating an overall higher content of proteins in the tissues since protein N is by far the largest N fraction constituting about 80–85% of the total N in green plant material (Mengel and Kirkby, 2001). The high protein content might be reflected in the photosynthetic capacity of the plants where NO3 fed plants had significantly lower Pmax rates than plants fed with NH4+ and NH4NO3. Other studies similarly found that NH4+ fed plants had a higher CO2 assimilation rate than NO3 fed plants, e.g. for Beta vulgaris (Raab and Terry, 1994), Rubus ideaus (Claussen and Lenz, 1999) and Phaseolus vulgaris (Bruck and Guo, 2006). The cause for the reduced photosynthetic capacity could be that assimilation of NO3 requires energy in the form of reducing power which is delivered as NADPH produced during the light reaction. A substantial part of the products of the electron chain may actually be used for NO3 assimilation, as the cost for reduction of 1 mol NO3 to NH4+ is 16 mol photons in illuminated green cells (Raven, 1985). However, when photosynthetic rates were expressed per unit Chl there was no significant difference between plants from the different treatments (data not shown). This indicates, that the biophysical function of the electron transport chain was

Fig. 1. Maximum N uptake rates per root dry weight (Vmax) of NH4+ and NO3 in Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days. Different letters indicate significant difference between treatments (P < 0.05). Values are means  S.D. (n = 5–6).

unaffected, and that the higher photosynthesis rate per leaf area in NH4+ fed plants was due to a restricted leaf expansion and subsequent higher Chl and enzyme densities (Claussen and Lenz, 1999; Guo et al., 2002). A restricted leaf expansion rate in NH4+ fed plants is often explained by a reduced cation uptake, or a less negative osmotic potential of leaves (Raab and Terry, 1995; WalchLiu et al., 2000). Previous studies have shown that the higher rates of photosynthesis in NH4+ fed plants compared to NO3 fed plants were accompanied by higher stomatal conductances (Høgh-Jensen and Schjoerring, 1997; Guo et al., 2002), but this was not observed in the present study on C. indica. Uptake rates of NO3 did not differ between NH4+ and NO3 fed plants indicating that the NO3 transporters in the plasma membranes of the root cells were not regulated by the presence of NO3 at the concentrations supplied in the uptake studies. The similar uptake rates of NO3 in plants regardless of treatment are in

Table 6 Results of two-way ANOVA for nitrate reductase activity in roots and leaves (plant fraction) of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days. Source of variance

Sum of squares

d.f.

Mean square

F-Ratio

P-Value

N-source Plant fraction N-source  plant fraction Residual

43.22 22.09 10.75

2 1 2

21.61 22.09 5.38

64.2 65.6 16.0

<0.0001 <0.0001 <0.0001

10.10

30

0.34

Fig. 2. Nitrate reductase activity (NRA) in roots and leaves of Canna indica grown at equimolar concentrations of different inorganic nitrogen forms (NO3, NH4NO3 or NH4+) for 23 days. Different letters indicate significant difference between treatments (P < 0.05). Values are means  S.D. (n = 5–6).

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148

agreement with a constitutive uptake system not regulated by N source or N concentration (Forde and Clarkson, 1999). In contrast, the Vmax for NH4+ uptake differed significantly between treatments and was highest in NH4+ fed plants and lowest in NO3 fed plants. This suggests that up-regulation of NH4+ uptake rates for NH4+ fed plants compared to NO3 fed plants is caused by a larger number of NH4+ transporters in the plasma membrane of the root cells. However, the higher uptake capacity could also be caused by a higher capacity to assimilate NH4+ in the roots of the NH4+ fed plants since NH4+ assimilation and NH4+ uptake seems to be coupled, and assimilation capacity affects transmembrane fluxes of NH4+ in plants (Loque and von Wiren, 2004). Tylova-Munzarova et al. (2005) found higher Vmax for NH4+ than NO3 in both NH4+ and NO3 fed P. australis and G. maxima indicating that these species are more adapted to wetlands than C. indica. In the aquatic floating fern, Salvinia natans, Jampeetong and Brix (2009) found that Vmax for NH4+ were constitutively high (590–792 mmol N g1 root DW h1) and not affected by N source, whereas Vmax for NO3 were significantly lower (42–125 mmol N g1 root DW h1) and affected by N source being highest in NO3 fed plants. Compared to the present study, Zhang et al. (2009) found higher Vmax of NH4+ and NO3 in C. indica; 238 and 167 mmol N g1 root DW h1, respectively, in a study where uptake rates were measured in younger plants after a starvation period of 2 days. This also indicated a preference for NH4+, but also shows that uptake rates are highly dependent on plant age and experimental conditions. The NH4+ fed plants generally had lower concentrations of the mineral cations Na+, Ca2+, Mg2+ and K+ in the plant tissues as has also been commonly observed for other species supplied with NH4+ as the N source (Britto and Kronzucker, 2002; Jampeetong and Brix, 2009). The lower uptake of cations in plants fed solely with NH4+ is probably one of the reasons why some plants suffer from NH4+ toxicity, since increased supply of Ca and K in some cases has been shown to alleviate NH4+ toxicity (Gerendas et al., 1997; Britto and Kronzucker, 2002; Roosta and Schjoerring, 2008). The growth repression of NH4+ fed Cucumis sativus at high concentrations (15 mM) of NH4+ or NO3, was accompanied by substantially lower concentrations of Ca2+ and Mg2+ in the plant tissues (Roosta and Schjoerring, 2007). Jampeetong and Brix (2009) also found lower concentrations of Ca2+, Mg2+ and K+ in Salvinia natans fed with NH4+ compared to NO3 fed plants. The changes in mineral composition observed in the present study for C. indica were, however, small and did probably not influence overall performance of the plants. Assimilation of NO3 can occur in both roots and shoots of plants (Andrews, 1986; Munzarova et al., 2006), and because of the energetic advantage of reducing NO3 in photosynthetic tissues, it has been suggested that the leaves may be the primary organ for NO3 reduction (Gojon et al., 1994). Furthermore, NO3 can readily be transported from the roots to the leaves via the xylem, as the NO3 ion does not have any toxic effects. In the present study, the NRA was clearly affected by N form in the culture solutions with almost no activity in NH4+ fed plants and high activities in NO3 fed plants. This is consistent with the common observation that the presence of NO3 induces NRA (Crawford et al., 1986; Gojon et al., 1994; Claussen and Lenz, 1999; Jampeetong and Brix, 2009). For plants supplied with both forms of N, a significant NRA was detected suggesting that C. indica actually take up and assimilate NO3 in the presence of NH4+. Furthermore, NH4+ fed plants had very low NRA in both roots and leaves whereas the plants supplied with NO3 alone or in combination with NH4+ had significantly higher NRA in leaves compared to roots. Similarly, Munzarova et al. (2006) found that P. australis had higher NRA in leaves compared to roots whereas G. maxima had similar NRA in leaves and roots. Both species had higher NRA when fed with NO3 compared to NH4+ treated plants. The NRA rates in leaves of NO3

147

fed plants were 19 and 37 mmol NO2 g1 DW h1 for P. australis and G. maxima, respectively, whereas in the present work NRA in leaves of NO3 fed C. indica was 22 mmol NO2 g1 DW h1. In a study of NRA in 14 aquatic macrophytes, Cedergreen and Madsen (2003) reported that plants growing in aquatic environments like streams and lakes generally had low NRA in roots and shoots (<2 mmol NO2 g1 DW h1) except the amphibious species Cardamine amara which had shoot NRA as high as 24 mmol NO2 g1 DW h1. The reduction of NO3 in the leaves is coupled to the light reactions which deliver electrons to the reduction. Thus, from an energetic point of view, photosynthetic tissue is more advantageous for NO3 reduction than respiratory root tissue when viewed on a whole plant basis (Raven, 1985). Slow growing plant species such as trees and woody scrubs typically reduce NO3 in roots whereas fast growing species tend to reduce a larger part of NO3 in the shoot even when grown at similar external NO3 concentrations as the slow growing species (Andrews, 1986; Gojon et al., 1994). Canna indica, as a fast growing species with high NRA in the leaves, fits with this generalisation. In conclusion, our results show that C. indica grows well with both NH4+ and NO3 as the sole N source. Canna indica did take up NH4+ at faster rates than NO3, and had higher contents of N in the tissue and higher photosynthetic rates when supplied with NH4+ suggesting a preference for NH4+. However, its N uptake characteristic with high and inducible NRA in the leaves corresponds to that of fast growing terrestrial species. Canna indica therefore seems to be relatively indifferent to inorganic N source, which together with its high growth rate contributes to explain the distribution of this species in flooded wetland soils as well as on terrestrial soils (De Kamer and Maas, 2008). The indifference to N nutrition also provides C. indica with the ability to have a high growth rate in periodically inundated areas with fluctuating and variable N sources depending on the soil redox conditions. The results further indicate that C. indica is suitable for use in constructed wetlands with horizontal subsurface flow where N mainly occurs as NH4+ and in unsaturated vertical flow CWs where NO3 predominates. Acknowledgements The study was funded by the Danish Natural Science Research Council. We thank Dr Brian Sorrell for helpful comments on the manuscript. References Andrews, M., 1986. The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ. 9, 511–519. Britto, D.T., Kronzucker, H.J., 2002. NH4+ toxicity in higher plants: a critical review. J. Plant Physiol. 159, 567–584. Brix, H., 1997. Do macrophytes play a role in constructed treatment wetlands? Water Sci. Technol. 35, 11–17. Brix, H., Schierup, H.H., 1989. The use of aquatic macrophytes in water-pollution control. Ambio 18, 100–107. Brix, H., Dyhr-Jensen, K., Lorenzen, B., 2002. Root-zone acidity and nitrogen source affects Typha latifolia L. growth and uptake kinetics of ammonium and nitrate. J. Exp. Bot. 53, 2441–2450. Brix, H., Koottatep, T., Laugesen, C.H., 2007. Wastewater treatment in tsunami affected areas of Thailand by constructed wetlands. Water Sci. Technol. 56, 69– 74. Bruck, H., Guo, S.W., 2006. Influence of N form on growth and photosynthesis of Phaseolus vulgaris L. plants. J. Plant Nutr. Soil Sci. 169, 849–856. Cedergreen, N., Madsen, T.V., 2003. Nitrate reductase activity in roots and shoots of aquatic macrophytes. Aquat. Bot. 76, 203–212. Chaillou, S., Vessey, J.K., Morotgaudry, J.F., Raper, C.D., Henry, L.T., Boutin, J.P., 1991. Expression of characteristics of ammonium nutrition as affected by pH of the root medium. J. Exp. Bot. 42, 189–196. Claussen, W., 2002. Growth, water use efficiency, and proline content of hydroponically grown tomato plants as affected by nitrogen source and nutrient concentration. Plant Soil 247, 199–209. Claussen, W., Lenz, F., 1999. Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and

148

D. Konnerup, H. Brix / Aquatic Botany 92 (2010) 142–148

glutamine synthetase in blueberry, raspberry and strawberry. Plant Soil 208, 95–102. Crawford, N.M., Campbell, W.H., Davis, R.W., 1986. Nitrate reductase from squash—cDNA cloning and nitrate regulation. Proc. Natl. Acad. Sci. U.S.A. 83, 8073–8076. De Kamer, H.M.V., Maas, P.J.M., 2008. The Cannaceae of the World. Blumea 53, 247– 318. Dyhr-Jensen, K., Brix, H., 1996. Effects of pH on ammonium uptake by Typha latifolia L. Plant Cell Environ. 19, 1431–1436. Forde, B.G., Clarkson, D.T., 1999. Nitrate and ammonium nutrition of plants: physiological and molecular perspectives. Adv. Bot. Res. 30, 1–90. Gerendas, J., Zhu, Z.J., Bendixen, R., Ratcliffe, R.G., Sattelmacher, B., 1997. Physiological and biochemical processes related to ammonium toxicity in higher plants. Z. Pflanzenerna¨hr. Bodenkd. 160, 239–251. Gojon, A., Plassard, C., Bussi, C., 1994. Root/shoot distribution of NO3 assimilaassimilation in herbaceous and woody species. In: Roy, J., Garnier, E. (Eds.), A Whole Plant Perspective on Carbon–Nitrogen Interactions. SPB Academic Publishing, Hague, pp. 131–148. Guo, S., Bruck, H., Sattelmacher, B., 2002. Effects of supplied nitrogen form on growth and water uptake of French bean (Phaseolus vulgaris L.) plants—nitrogen form and water uptake. Plant Soil 239, 267–275. Guo, S., Zhou, Y., Shen, Q., Zhang, F., 2007. Effect of ammonium and nitrate nutrition on some physiological processes in higher plants—growth, photosynthesis, photorespiration, and water relations. Plant Biol. 9, 21–29. Høgh-Jensen, H., Schjoerring, J.K., 1997. Effects of drought and inorganic N form on nitrogen fixation and carbon isotope discrimination in Trifolium repens. Plant Physiol. Biochem. 35, 55–62. Jampeetong, A., Brix, H., 2009. Nitrogen nutrition of Salvinia natans: effects of inorganic nitrogen form on growth, morphology, nitrate reductase activity and uptake kinetics of ammonium and nitrate. Aquat. Bot. 90, 67–73. Kadlec, R.H., Wallace, S.D., 2008. Treatment Wetlands. CRC Press, Boca Raton, FL. Konnerup, D., Koottatep, T., Brix, H., 2009. Treatment of domestic wastewater in tropical, subsurface flow constructed wetlands planted with Canna and Heliconia. Ecol. Eng. 35, 248–257. Kronzucker, H.J., Siddiqi, M.Y., Glass, A.D.M., 1997. Conifer root discrimination against soil nitrate and the ecology of forest succession. Nature 385, 59–61. Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids—pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–382. Loque, D., von Wiren, N., 2004. Regulatory levels for the transport of ammonium in plant roots. J. Exp. Bot. 55, 1293–1305. Marschner, H., 1995. Mineral Nutrition of Higher Plants. Academic Press, London. Mengel, K., Kirkby, E.A., 2001. Principles of Plant Nutrition. Kluwer Academic Publishers, Dordrecht.

Munzarova, E., Lorenzen, B., Brix, H., Vojtiskova, L., Votrubova, O., 2006. Effect of NH4+/NO3 availability on nitrate reductase activity and nitrogen accumulation in wetland helophytes Phragmites australis and Glyceria maxima. Environ. Exp. Bot. 55, 49–60. Nijburg, J.W., Laanbroek, H.J., 1997. The fate of N-15-nitrate in healthy and declining Phragmites australis stands. Microb. Ecol. 34, 254–262. Oscarson, P., Ingemarsson, B., Ugglas, M.A., Larsson, C.M., 1988. Characteristics of NO3 Uptake in Lemna and Pisum. Plant Soil 111, 203–205. Raab, T.K., Terry, N., 1994. Nitrogen source regulation of growth and photosynthesis in Beta Vulgaris L. Plant Physiol. 105, 1159–1166. Raab, T.K., Terry, N., 1995. Carbon, nitrogen and nutrient interactions in Beta vulgaris L. as influenced by nitrogen source, NO3 versus NH4+ Plant Physiol. 107, 575– 584. Raven, J.A., 1985. Regulation of pH and generation of osmolarity in vascular plants— a cost-benefit analysis in relation to efficiency of use of energy, nitrogen and water. New Phytol. 101, 25–77. Romero, J.A., Brix, H., Comin, F.A., 1999. Interactive effects of N and P on growth, nutrient allocation and NH4 uptake kinetics by Phragmites australis. Aquat. Bot. 64, 369–380. Roosta, H.R., Schjoerring, J.K., 2007. Effects of ammonium toxicity on nitrogen metabolism and elemental profile of cucumber plants. J. Plant Nutr. 30, 1933–1951. Roosta, H.R., Schjoerring, J.K., 2008. Effects of nitrate and potassium on ammonium toxicity in cucumber plants. J. Plant Nutr. 31, 1270–1283. Scheible, W.R., Lauerer, M., Schulze, E.D., Caboche, M., Stitt, M., 1997. Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco. Plant J. 11, 671–691. Strojny, Z., 1999. Effect of nutrient solution concentration and NH4:NO3 ratio on Maranta growth. Sci. Hortic. 80, 105–112. Tylova-Munzarova, E., Lorenzen, B., Brix, H., Votrubova, O., 2005. The effects of NH4+ and NO3 on growth, resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima. Aquat. Bot. 81, 326–342. Walch-Liu, P., Neumann, G., Bangerth, F., Engels, C., 2000. Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J. Exp. Bot. 51, 227–237. Zhang, Z., Rengel, Z., Meney, K., 2009. Kinetics of ammonium, nitrate and phosphorus uptake by Canna indica and Schoenoplectus validus. Aquat. Bot. 91, 71–74. Zou, C.Q., Wang, X.F., Wang, Z.Y., Zhang, F.S., 2005. Potassium and nitrogen distribution pattern and growth of flue-cured tobacco seedlings influenced by nitrogen form and calcium carbonate in hydroponic culture. J. Plant Nutr. 28, 2145–2157. Zurita, F., De Anda, J., Belmont, M.A., 2009. Treatment of domestic wastewater and production of commercial flowers in vertical and horizontal subsurface-flow constructed wetlands. Ecol. Eng. 35, 861–869.