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Spatial distribution of ammonium and nitrate fluxes along roots of wetland plants
Plant Science 173 (2007) 240–246 www.elsevier.com/locate/plantsci
Spatial distribution of ammonium and nitrate fluxes along roots of wetland plants Yun Ying Fang a,b, Olga Babourina b,*, Zed Rengel b, Xiao E Yang a, Pei Min Pu c b
a College of Environmental and Resource Sciences, Zhejiang University, 310029 Hangzhou, China Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia c Nanjing Institute of Geography & Limnology, CAS, Nanjing 210008, China
Received 12 February 2007; received in revised form 15 May 2007; accepted 21 May 2007 Available online 29 May 2007
Abstract Nothing is known about nutrient fluxes along the roots of floating and submerged macrophytes that may be used for removing nutrients from eutrophicated water systems. We have used ion-sensitive microelectrodes to measure fluxes of NH4+, NO3 and H+ along the root apices. One floating (Azolla spp.) and three submerged plant species (Vallisneria natans Lour. Hara; Bacopa monnieri L. Pennell; and Ludwigia repens J.R. Forst) were tested. Ion fluxes showed a specific pattern linked to root zones in all four species. The highest influx of all three ions was found in the meristem zone of B. monnieri, V. natans and L. repens. B. monnieri had the greatest capacity to acidify the surrounding medium. In the four species studied, there was a consistent negative relationship between the fluxes of NO3 and H+ measured simultaneously along the root and a positive relationship between H+ and NH4+ fluxes. When NH4+ and NO3 were both present in the bathing medium, the meristem zone had the largest capacity for net NH4+ uptake, whereas the elongation zone showed the highest net NO3 uptake. In the short-term experiments, Azolla spp. had preference for NO3 uptake when NO3 was supplied as a sole source of nitrogen, whereas L. repens required both nitrogen forms in the medium for net nitrogen uptake. When NO3 and NH4+ fluxes were summed, L. repens had the largest and V. repens the smallest nitrogen accumulation capacity. Therefore, for industrial purposes, when plants are used for removing N from eutrophicated water, plants species should be selected according to their preferences for different N forms. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Bacopa monnieri; Vallisneria natans; Azolla spp.; Ludwigia repens; Ammonium; Nitrate
1. Introduction Ecological engineering offers a simple, cheap and energyefficient method of treating polluted water and wastewater. Aquatic macrophytes can take up excessive nutrients and also play a crucial role in creating a favourable environment for a variety of chemical, biological and physical processes that contribute to the nutrient removal and degradation of organic compounds [1,2]. The main research on aquatic plants has been focused on comparing actual nutrient uptake and improving the nutrient uptake potential from polluted water and wastewater by Abbreviation: N, nitrogen * Corresponding author. Tel.: +61 8 6488 2846; fax: +61 8 6488 1050. E-mail address: [email protected] (O. Babourina). 0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2007.05.006
different species across time and space [3]. However, there is little knowledge about the capacity of roots of aquatic plants to absorb ammonium (NH4+) and nitrate (NO3 ). For efficient nitrogen (N) removal in ecological engineering, it is important to quantify the preferences of aquatic plants for various N forms and to characterise the relationship between uptake rates of the two mineral forms of N (NH4+ and NO3 ). Given that ion uptake by various parts of the root is variable [4,5], the profiles of H+, NH4+ and NO3 fluxes were measured non-invasively along roots of four aquatic plants by the Microelectrode Ion Flux Estimation (MIFE) technique. To date the root profiles of both NH4+ and NO3 fluxes were reported for only four plant species: barley [6], rice [7], maize [7,8] and Eucalyptus nitens [4]. In all earlier studies ion fluxes demonstrated high spatial and temporal variability, especially in the elongation zone of the root. Moreover, NH4+ fluxes
Y.Y. Fang et al. / Plant Science 173 (2007) 240–246
measured for the same plant species (e.g. maize) were not characterised in the same way [7,8]. On the other hand, E. nitens profiles were obtained for the mature zone of the root only [4]. In our recent studies we reported spatial profiles of H+, NH4+ and NO3 fluxes in an aquatic plant Landoltia punctata [9]. Reviewing literature available to date, it is not possible to draw clear conclusions and predict NH4+ and NO3 flux distribution along different zones of the plant root. There are some indications that the meristem zone prefers NH4+ to NO3 for uptake when both nitrogen forms are supplied, however, significant difference was found only for rice [8]. Interestingly, the meristem zone has been characterised as the root zone with the highest H+ influx [5]. Therefore, if high NH4+ uptake is also assigned to the meristem zone, it will be contradictory to a hypothesis, which considers acidification of the rhizosphere as the main reason of NH4+ toxicity [10]. In the present study, we used the MIFE technique to map net NH4+, NO3 and H+ fluxes along the primary roots of wetland plants to clarify the relationship between H+ and NH4+ fluxes and plant preferences for N forms. 2. Materials and methods 2.1. Plant materials One floating and three submerged plant species were tested: (i) Azolla spp., floating fern (family Salviniaceae) containing nitrogen-fixing blue-green algae, extremely fast-growing; (ii) Vallisneria natans (Lour.) Hara. (family Hydrocharitaceae), a perennial submerged plant with a wide geographical range, can grow in different sediments in either nutrient-poor or nutrient-rich freshwater systems; (iii) Bacopa monnieri (L.) Pennell (family Scrophulariaceae) (herb-of-grace, water hyssop), perennial succulent creeping herb growing in wetlands and on muddy shores; and (iv) Ludwigia repens J.R. Forst. (family Onagraceae), creeping primrose willow, mostly submerged perennial herb, commonly growing in shallow marshy areas. Plants were acquired from a local commercial supplier in Perth, Western Australia, and cultivated in a glasshouse at The University of WA. The plants were cultured in 50 L opaque plastic containers on a modified Hoagland nutrient solution before treatments. The solution contained (in mM): NH4+ 0.143; NO3 0.143; Ca2+ 0.05; Mg2+ 0.025; Na+ 0.56; K+ 0.025; H2PO4 0.032; Cl 0.125; SO42 , 0.05 and CO3 0.2. Positions of root apex, meristem location and expansion zone were determined under an optical microscope. 2.2. Ion flux measurements Ion fluxes were measured non-invasively using the MIFE1 system (University of Tasmania, Hobart, Australia) as described by Newman [5]. Electrodes were pulled from borosilicate glass capillaries (GC150-10, Harvard Apparatus, Kent, UK), dried at 230 8C for about 5 h, and silanised with tributylchlorosilane (90765, Fluka Chemicals). The tips of dried and cooled electrode blanks were broken to a diameter of 5–10 mm and then back-filled with appropriate solutions. Back-
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filling solutions were 15 mM NaCl and 40 mM KH2PO4 for the hydrogen electrode, 500 mM NH4Cl for the ammonium, and 500 mM KNO3 and 100 mM KCl for the nitrate electrode. Immediately after back-filling, the electrode apices were frontfilled with commercially available ionophore cocktails for measuring hydrogen (#95297, Fluka) and ammonium (# 09879, Fluka). The nitrate sensor contained 0.5% (w/v) methyltridodecylammonium nitrate (MTDDA NO3 ), 0.084% (w/v) methyltriphenylphosphonium bromide (MTPPB) and 99.4% (v/v) n-phenyloctyl ether (NPOE) [11,12]. A reference electrode was fabricated in a similar way from a borosilicate glass capillary and filled with 0.1 M KCl in 2% (w/v) agar. Electrodes were calibrated against a range of standards (pH from 5.3 to 7.2, NH4+ and NO3 from 0.1 to 10 mM). Electrodes with responses less than 50 mV per decade were discarded. 2.3. Experimental procedures The roots were measured in Petri dishes placed on an inverted microscope. In the experiments to determine ammonium, nitrate and proton fluxes along the roots of aquatic plants, the bathing solution contained (in mM): NH4NO3 1.0; CaCl2 0.05; MgSO4 0.025; NaCl 0.56; K2SO4 0.025; NaH2PO4 0.032 and NaHCO3 0.2; pH 6.8–7.0. Thirty minutes after the pre-treatment with basal solution, measurements of ion fluxes commenced at 60–120 mm from the root surface. In our earlier studies we have shown that in media with NO3 , ion fluxes become stable 30 min after increase in NH4+ concentration [13]. In the mapping experiment, 3–4 replicate plants were measured and averaged. To cope with the oscillation pattern of ion fluxes in the elongation zone we measured meristem and elongation zones several times and the median value of ion fluxes for each plant was used for averaging between plants. Experiments were performed at 20–22 8C under standard laboratory lighting. Fluxes of H+, NH4+ and NO3 were measured at each root position simultaneously (see Fig. 2A–C). For estimation of plant preferences for NH4+ or NO3 supply, measurements were done on the same root position after 30 min of exposure to solution containing 1.0 mM of either NH4Cl, KNO3 or NH4NO3 in random order, pH 6.5–7.0. Each measurement took 5 mins (three root positions: apex and two points at the elongation zone). The Petri dish was then flushed with new solution containing the appropriate N source (NH4NO3 and NH4Cl). Flux measurements were performed 30 min after the transfer for plant adjustment to a new solution. Means and standard errors were calculated for 4–6 plants propagated and measured separately. Significant difference between means was assessed by Genstat 8th edition (VSN International Ltd., Hemel Hempstead, UK) for ANOVA analysis. 3. Results 3.1. H+ fluxes and pH profiles along the roots of aquatic plants Fluxes of H+, NO3 and NH4+ were measured at various positions (root apex, meristem, distal and proximal elongation
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Fig. 1. Root morphology under optical microscope. (A) Azolla spp.; (B) Bacopa monnieri; (C) Vallisneria natans; (D) Ludwigia repens.
zones) along the roots of four wetland plant species (Fig. 1A– D). Since Azolla spp. had a distinct root hair zone, fluxes were measured in that zone as well. The H+ fluxes demonstrated a highly specific pattern at different root zones (Fig. 2). The meristem zone was characterised as the zone with the highest H+ influx or the lowest efflux. In Azolla spp. and L. repens, the rate of H+ fluxes at the root apex was very low, around 0 nmol m 2 s 1. The apex and the meristem zone of V. natans roots were characterised by high H+ influx (around 60 nmol m 2 s 1). The distal and proximal elongation zone of Azolla spp., B. monnieri and L. repens showed H+ efflux in absolute values (Fig. 2). B. monnieri had the largest magnitude of H+ fluxes and the highest ability to
Table 1 Net fluxes (nmol m or NH4NO3) Species
2
acidify the basal media to a greater extent than other plant species tested (1.2 pH units in 2 h). 3.2. Mapping of NH4+ and NO3 fluxes along the roots of aquatic plants When NH4+ and NO3 were supplied together in B. monnieri, V. natans and L. repens, the highest net NH4+ uptake was measured in the meristem zone, decreasing at more mature regions (Fig. 2B). Specifically, the biggest difference between the high NH4+ uptake rate at the meristem zone and the low uptake rate at more mature regions was recorded for V. natans
s 1) of NH4+ and NO3 at the same points of the different root zones of four wetland species exposed to different media (1.0 mM NH4Cl, KNO3 Distance from tip (mm)
1.0 mM NH4Cl
1.0 mM KNO3
1.0 mM NH4NO3
NH4+
NO3 flux
NH4+ flux
flux
Azolla spp.
0 1200 2400
50 37ef 8 49e 106 19f
274 54cd 398 17c 296 61cd
Bacopa monnieri
0 1200 2400
81 42de 48 21e 47 28e
172 38d 255 49cd 273 86cd
Vallisneria natans
0 1200 2400
66 23ef 9 35e 3 37e
Ludwigia repens
0 1200 2400
88 18de 74 25de 68 55e
66 54def 23 43ef 38 32e
NO3 flux
NH4+ + NO3 flux
112 61f 47 71e 177 92d
178 76f 24 34e 215 65cd
250 53cd 108 47d 129 61de
14 23e 109 27d 196 29d
264 50cd 217 62cd 325 54c
74 25de 46 10e 74 26de
600 40 b 57 39e 18 39e
347 54f 37 75e 26 35e
253 64cd 20 68e 8 29e
30 28ef 8 43e 2 27e
372 56c 23 30e 74 40e
36 55e 665 43b 1015 53a
408 81bc 688 46b 1089 52a
Negative net values represent ion efflux, and positive values represent ion influx. Values are mean S.E.M. (n = 4–6). Different letters indicate significant differences ( p < 0.05) estimated by ANOVA.
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(from around 600 nmol m 2 s 1 down to zero), followed by L. repens (from around 400 nmol m 2 s 1 to zero). B. monnieri maintained the highest positive flux of NH4+ in the distal elongation zone among the species studied. Azolla spp.
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demonstrated the lowest variation in NH4+ spatial distribution along its root. In all four plant species studied, NO3 fluxes were the lowest at the root apex (Fig. 2C) and increased towards more mature
Fig. 2. Net H+ (A), NH4+ (B) and NO3 (C) fluxes along various root parts as noted in the top bars: root apex (hatched), meristem zone (black), distal elongation zone (white), proximal elongation zone (grey) and root hair zone (vertical lines). Flux measurement were taken while roots were exposed to the basal solution containing (in mM) NH4NO3 1.0; CaCl2 0.05; MgSO4 0.025; NaCl 0.56; K2SO4 0.025; NaH2PO4 0.032; and NaHCO3 0.2.
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Fig. 2. (Continued ).
regions peaking immediately behind the root apex. The relatively constant NO3 flux in the proximal elongation zone was the lowest in Valisneria natans (around 0 nmol m 2 s 1) and the highest in L. repens (around 1000 nmol m 2 s 1). 3.3. NH4+ and NO3 fluxes in relation to their availability A comparison was made between the rates of NH4+ and NO3 fluxes with different N source (NH4Cl, KNO3 or NH4NO3 ) in the bathing medium (Table 1). When both forms of N were available (1 mM NH4NO3 ), L. repens preferred NO3 for uptake at both points of the elongation zone (Table 1). All species except L. repens took up N effectively when NO3 was the sole source. Even when NH4+ was the only source of N (1 m NH4Cl), Azolla spp. did not switch on the NH4+ uptake system along the root segment monitored. A requirement for presence of both N forms in the bathing media was observed for L. repens; it accumulated NH4+ and NO3 in 1 mM NH4NO3, whereas it excluded NO3 in 1 mM KNO3 and NH4+ in 1.0 mM NH4Cl. When NH4+ and NO3 fluxes were summed in media containing1 mM NH4NO3 , L. repens had the largest N accumulation capacity, whereas Azolla spp. and V. repens took up N at a lower rate. 4. Discussion In this study, it was found that different aquatic plant species had different capacity to acidify external media: B. monnieri had the highest absolute values of H+ efflux (Fig. 2), showing a high capacity to acidify the media: it acidified and maintained
the pH of the bathing media at pH 5.4–5.6 (data not shown). This finding explains this species plasticity in regards to high external pH: B. monnieri usually is found in lakes with high pH [14]. The H+ fluxes in the media containing different forms of N were similar (data not shown), which is in accordance with studies on other plant species. In Lolium perenne and Trifolium repens, the H+ flux was generally unaffected by the concentration of external NO3 and NH4+ and did not correlate with the rates of N absorption [15]. Generally, the root apex, meristematic and apical elongation regions demonstrate net H+ influx in most plant species studied, with resulting alkalization of the adjacent medium [5]. Similarly, in our study all plants had the highest net H+ influx or the lowest H+ efflux at the root apex and meristem zones. However, the absolute values of H+ fluxes for three aquatic species, apart from V. natans, (Fig. 2) were relatively small compared with other studies [4,8,16]. Such small H+ flux could be explained by the high pH of our bathing solution (pH 6.5–7.0), compared to that in other studies (pH 5.5–6.0). However, this pH is closer to that of native ecosystems (pH 7–8; Fang, unpublished). As expected, there was a consistent relationship between the fluxes of NH4+ and H+ measured simultaneously at each root position for all plants studied: Azolla spp. (r = 0.57); B. monnieri (r = 0.73); V. natans (r = 0.95); and L. repens (r = 0.85). Higher NH4+ uptake rate coincided with higher H+ uptake and increased pH in the root apex zone (data not shown). Therefore, our results provide additional information to a hypothesis that NH4+ toxicity is explained by acidification of the external media during NH4+ supply as the sole N source,
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showing that the observed in other studies [17] acidification is not directly linked to H+ extrusion during NH4+ uptake. The high correlation between NH4+ and H+ fluxes are in agreement with studies performed on eucalypt roots where authors used the same MIFE technique [4], which calculates ion fluxes more comprehensively, analysing diffusion rate of ions at the particular area of the root [5]. However, in the majority of other studies H+ flux was estimated as a change of the bulk pH, which could reflect different reasons including a change in the concentration of other ions (e.g. carbonate). Also, kinetics analysis of NH4+ uptake, estimated by measurements of plasma membrane potential, allowed authors to suggest H+–NH4+ symport in rice roots [18]. In addition to different techniques used for studying the interdependence of NH4+ and H+ transport, other factors could be responsible for the contradicting results, such as the time of plant exposure to different N sources or the different experimental setup (‘‘disturbed’’ or ‘‘undisturbed’’ conditions). From our study, it is plausible to suggest that different root zones prefer different forms of N, when both forms of N are available in the surrounding media. A larger net influx of NH4+ was observed in the meristem zone in comparison with the elongation region (Fig. 2B, Table 1). Similar results were observed for rice when plants were exposed to NH4+ and NO3 simultaneously [8]. For maize roots these researchers also observed a small decline in net NH4+ uptake in the elongation zone compared with the root apex and meristem zone. Nitrate uptake was higher at the elongation zone than in other root regions in all four species studied (Fig. 2C, Table 1). Similarly, Siebrecht et al. [19] found that NO3 uptake into the apex of barley roots was only half of that into the older root zones. When both NH4+ and NO3 were supplied together, the root apices exhibited higher net influx (or smaller net efflux) of NH4+ than NO3 , but the magnitude differed among species (Table 1). Net NH4+ uptake was 15-fold greater than net NO3 uptake in B. monnieri and 10-fold greater in L. repens. Similarly, net NH4+ uptake was 2-fold greater than net NO3 uptake in the maize root apex zone [8], and 3-fold greater in rice roots [7]. Several explanations can be suggested for the observed behaviour in this clearly defined root zone, where plants are taking NH4+ at a higher rate than NO3 . The first explanation suggested based on root morphology: different root tissues demand different amounts of NH4+ and NO3 and the meristem zone requires a higher amount of NH4+ for protein synthesis [8]. The second explanation is based on the different expression and activities of transport systems for both NH4+ and NO3 in different root zones. Net NH4+ and NO3 uptake can be mediated by high-affinity transporters as well as various low-affinity transporters, and can be reversed by NH4+ and NO3 efflux systems. To date, several transporters have been cloned: high affinity NH4+ transporters from the ammonium transporters (AMT) family and NO3 transporters from nitrate– nitrite (NNP) and peptide transporters (PTR) [20,21]. However, there is no information on their expression pattern along the root apex. No specific transporters that function as NH4+ or NO3 efflux transporters have been cloned yet.
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Our study demonstrated again that different plant species require different forms of N [21]. From four plants studied, two (B. monnieri and Azolla spp.) had preference for NO3 , whereas both N forms were required by L. repens for N uptake (Table 1). The limited ability of Azolla spp. plants to take up NH4+, which was observed in our study as NH4+ efflux in mono-NH4+ or mixed (NH4+ and NO3 ) media, is consistent with their biological requirements. Being a symbiont with Anabaena azollae, a nitrogen fixing cyanobacteria, Azolla plants are usually well supplied with NH4+ [22]. Plant preference for different forms of N is influenced by environmental factors such as root- or air-temperature, aeration, solution pH, composition of the culture solution, water stress and high concentration of salts in the root zone, and also by the plant growth stage and its ability to form a symbiosis with bacteria or fungi [22–24]. It could be suggested that these different N-uptake requirements depend on different growth rates or phenological phases of the plants studied. The precise mechanisms underpinning NH4+/NO3 interactions in aquatic species should be further investigated. Therefore, for industrial purposes, when plants are used for removing N from eutrophicated water, plants species should be selected according to their preferences for different N forms.
Acknowledgements We are grateful to Dr. Ian Newman, the University of Tasmania, who kindly lent us the MIFE system for selective ion measurements. Root pictures were taken in the Centre for Microscopy and Microanalysis, the University of Western Australia. The project was supported by the China Fund (administered by the Australian Department of Education, Science and Training).
References [1] T. Gumbricht, Nutrient removal processes in freshwater submersed macrophyte systems, Ecol. Eng. 2 (1993) 1–30. [2] Y.X. Chong, H.Y. Hu, Y. Qian, Advances in utilization of macrophytes in water pollution control, Tech. Equip. Env. Pol. Contr. 4 (2004) 36–40, in Chinese. [3] F. Wu, H. Qing, G. Wan, Regeneration of N, P and Si near the sediment/ water interface of lakes from Southwestern China Plateau, Water Res. 35 (2001) 1334–1337. [4] T.P. Garnett, S.N. Shabala, P.J. Smethurst, I.A. Newman, Simultaneous measurement of ammonium, nitrate and proton fluxes along the length of eucalypt roots, Plant Soil 236 (2001) 55–62. [5] I.A. Newman, Ion transport in roots, measurement of fluxes using ionselective microelectrodes to characterize transporter function, Plant Cell Environ. 24 (2001) 1–14. [6] G.H. Henriksen, D.R. Raman, L.P. Walker, R.M. Spanswick, Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique, Plant Physiol. 99 (1992) 734–747. [7] T.D. Colmer, A.J. Bloom, A comparison of NO3 and NH4+ net fluxes along roots of rice and maize, Plant Cell Environ. 21 (1998) 240–246. [8] A.R. Taylor, A.J. Bloom, Ammonium, nitrate, and proton fluxes along the maize root, Plant Cell Environ. 21 (1998) 1255–1263.
246
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[9] Y.Y. Fang, O. Babourina, Z. Rengel, X.E. Yang, P.M. Pu, Ammonium and nitrate uptake by the floating plant Landoltia punctata, Ann. Bot. 99 (2007) 365–370. [10] J. Gerenda´s, Z. Zhu, R. Bendixen, R.G. Ratcliffe, B. Sattelmacher, Physiological and biochemical processes related to ammonium toxicity in higher plants, Z. Pflanzen. Boden. 160 (1997) 239–251. [11] R.G. Zhen, S.J. Smith, A.J. Miller, A comparison of nitrate-selective microelectrodes made with different nitrate sensors and the measurement of intracellular nitrate activities in cells of excised barley roots, J. Exp. Bot. 43 (1992) 131–138. [12] C. Plassard, A. Gue´rin-Laguette, A.A. Ve´ry, V. Casarin, J.B. Thibaud, Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effect of ectomycorrhizal symbiosis, Plant Cell Environ. 25 (2002) 75–84. [13] O. Babourina, K. Voltchanskii, B. McGann, I. Newman, Z. Rengel, Nitrate supply affects ammonium transport in canola roots, J. Exp. Bot. (2007) 651–658. [14] M.V. Hoyer, D.E. Canfield, C.A. Horsbourgh, K. Brown, Florida Freshwater Plants: A Handbook of Common Aquatic Plants in Florida Lakes, University of Florida, Institute of Food and Agricultural Sciences, Gainesville, FL, 1996. [15] H. Høgh-Jensen, B. Wollenweber, J.K. Schjoerring, Kinetics of nitrate and ammonium absorption and accompanying H+ fluxes in roots of Lolium perenne L. and N2-fixing Trifolium repens L., Plant Cell Environ. 20 (1997) 1184–1192.
[16] T.P. Garnett, S.N. Shabala, P.J. Smethurst, I.A. Newman, Kinetics of ammonium and nitrate uptake by eucalypt roots and associated proton fluxes measured using ion selective microelectrodes, Funct. Plant Biol. 30 (2003) 1165–1176. [17] A.L. Miller, G.N. Smith, J.A. Raven, N.A. Gow, Ion currents and the nitrogen status of roots of Hordeum vulgare and non-nodulated Trifolium repens, Plant Cell Environ. 14 (1991) 559–567. [18] M.Y. Wang, A.D.M. Glass, J.E. Shaff, L.V. Kochian, Ammonium uptake by rice roots. III. Electrophysiology, Plant Physiol. 104 (1993) 899–906. [19] S. Siebrecht, G. Ma¨ck, R. Tischner, Function and contribution of the root apex in the induction of NO3 uptake along the barley root axis, J. Exp. Bot. 46 (1995) 1669–1676. [20] B.G. Forde, Nitrate transporters in plants: structure, function and regulation, Biochem. Biophys. Acta 1465 (2000) 219–235. [21] D. Loque, N. von Wiren, Regulatory levels for the transport of ammonium in plant root, J. Exp. Bot. 55 (2004) 1293–1305. [22] J.C. Meeks, N.A. Steinberg, C.S. Enderlin, C.M. Joseph, G.A. Peters, Azolla–Anabaena relationship. XIII. Fixation of [13N]N2, Plant Physiol. 84 (1987) 883–886. [23] H. Ikeda, Utilization of nitrogen by vegetable crops, Jap. Agricult. Res. Quart. 25 (1991) 117–124. [24] T.P. Garnett, P.J. Smethurst, Ammonium and nitrate uptake by Eucalyptus nitens, effect of pH and temperature, Plant Soil 214 (1999) 133–140.
Spatial distribution of ammonium and nitrate fluxes along roots of wetland plants Yun Ying Fang a,b, Olga Babourina b,*, Zed Rengel b, Xiao E Yang a, Pei Min Pu c b
a College of Environmental and Resource Sciences, Zhejiang University, 310029 Hangzhou, China Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia c Nanjing Institute of Geography & Limnology, CAS, Nanjing 210008, China
Received 12 February 2007; received in revised form 15 May 2007; accepted 21 May 2007 Available online 29 May 2007
Abstract Nothing is known about nutrient fluxes along the roots of floating and submerged macrophytes that may be used for removing nutrients from eutrophicated water systems. We have used ion-sensitive microelectrodes to measure fluxes of NH4+, NO3 and H+ along the root apices. One floating (Azolla spp.) and three submerged plant species (Vallisneria natans Lour. Hara; Bacopa monnieri L. Pennell; and Ludwigia repens J.R. Forst) were tested. Ion fluxes showed a specific pattern linked to root zones in all four species. The highest influx of all three ions was found in the meristem zone of B. monnieri, V. natans and L. repens. B. monnieri had the greatest capacity to acidify the surrounding medium. In the four species studied, there was a consistent negative relationship between the fluxes of NO3 and H+ measured simultaneously along the root and a positive relationship between H+ and NH4+ fluxes. When NH4+ and NO3 were both present in the bathing medium, the meristem zone had the largest capacity for net NH4+ uptake, whereas the elongation zone showed the highest net NO3 uptake. In the short-term experiments, Azolla spp. had preference for NO3 uptake when NO3 was supplied as a sole source of nitrogen, whereas L. repens required both nitrogen forms in the medium for net nitrogen uptake. When NO3 and NH4+ fluxes were summed, L. repens had the largest and V. repens the smallest nitrogen accumulation capacity. Therefore, for industrial purposes, when plants are used for removing N from eutrophicated water, plants species should be selected according to their preferences for different N forms. # 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Bacopa monnieri; Vallisneria natans; Azolla spp.; Ludwigia repens; Ammonium; Nitrate
1. Introduction Ecological engineering offers a simple, cheap and energyefficient method of treating polluted water and wastewater. Aquatic macrophytes can take up excessive nutrients and also play a crucial role in creating a favourable environment for a variety of chemical, biological and physical processes that contribute to the nutrient removal and degradation of organic compounds [1,2]. The main research on aquatic plants has been focused on comparing actual nutrient uptake and improving the nutrient uptake potential from polluted water and wastewater by Abbreviation: N, nitrogen * Corresponding author. Tel.: +61 8 6488 2846; fax: +61 8 6488 1050. E-mail address: [email protected] (O. Babourina). 0168-9452/$ – see front matter # 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2007.05.006
different species across time and space [3]. However, there is little knowledge about the capacity of roots of aquatic plants to absorb ammonium (NH4+) and nitrate (NO3 ). For efficient nitrogen (N) removal in ecological engineering, it is important to quantify the preferences of aquatic plants for various N forms and to characterise the relationship between uptake rates of the two mineral forms of N (NH4+ and NO3 ). Given that ion uptake by various parts of the root is variable [4,5], the profiles of H+, NH4+ and NO3 fluxes were measured non-invasively along roots of four aquatic plants by the Microelectrode Ion Flux Estimation (MIFE) technique. To date the root profiles of both NH4+ and NO3 fluxes were reported for only four plant species: barley [6], rice [7], maize [7,8] and Eucalyptus nitens [4]. In all earlier studies ion fluxes demonstrated high spatial and temporal variability, especially in the elongation zone of the root. Moreover, NH4+ fluxes
Y.Y. Fang et al. / Plant Science 173 (2007) 240–246
measured for the same plant species (e.g. maize) were not characterised in the same way [7,8]. On the other hand, E. nitens profiles were obtained for the mature zone of the root only [4]. In our recent studies we reported spatial profiles of H+, NH4+ and NO3 fluxes in an aquatic plant Landoltia punctata [9]. Reviewing literature available to date, it is not possible to draw clear conclusions and predict NH4+ and NO3 flux distribution along different zones of the plant root. There are some indications that the meristem zone prefers NH4+ to NO3 for uptake when both nitrogen forms are supplied, however, significant difference was found only for rice [8]. Interestingly, the meristem zone has been characterised as the root zone with the highest H+ influx [5]. Therefore, if high NH4+ uptake is also assigned to the meristem zone, it will be contradictory to a hypothesis, which considers acidification of the rhizosphere as the main reason of NH4+ toxicity [10]. In the present study, we used the MIFE technique to map net NH4+, NO3 and H+ fluxes along the primary roots of wetland plants to clarify the relationship between H+ and NH4+ fluxes and plant preferences for N forms. 2. Materials and methods 2.1. Plant materials One floating and three submerged plant species were tested: (i) Azolla spp., floating fern (family Salviniaceae) containing nitrogen-fixing blue-green algae, extremely fast-growing; (ii) Vallisneria natans (Lour.) Hara. (family Hydrocharitaceae), a perennial submerged plant with a wide geographical range, can grow in different sediments in either nutrient-poor or nutrient-rich freshwater systems; (iii) Bacopa monnieri (L.) Pennell (family Scrophulariaceae) (herb-of-grace, water hyssop), perennial succulent creeping herb growing in wetlands and on muddy shores; and (iv) Ludwigia repens J.R. Forst. (family Onagraceae), creeping primrose willow, mostly submerged perennial herb, commonly growing in shallow marshy areas. Plants were acquired from a local commercial supplier in Perth, Western Australia, and cultivated in a glasshouse at The University of WA. The plants were cultured in 50 L opaque plastic containers on a modified Hoagland nutrient solution before treatments. The solution contained (in mM): NH4+ 0.143; NO3 0.143; Ca2+ 0.05; Mg2+ 0.025; Na+ 0.56; K+ 0.025; H2PO4 0.032; Cl 0.125; SO42 , 0.05 and CO3 0.2. Positions of root apex, meristem location and expansion zone were determined under an optical microscope. 2.2. Ion flux measurements Ion fluxes were measured non-invasively using the MIFE1 system (University of Tasmania, Hobart, Australia) as described by Newman [5]. Electrodes were pulled from borosilicate glass capillaries (GC150-10, Harvard Apparatus, Kent, UK), dried at 230 8C for about 5 h, and silanised with tributylchlorosilane (90765, Fluka Chemicals). The tips of dried and cooled electrode blanks were broken to a diameter of 5–10 mm and then back-filled with appropriate solutions. Back-
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filling solutions were 15 mM NaCl and 40 mM KH2PO4 for the hydrogen electrode, 500 mM NH4Cl for the ammonium, and 500 mM KNO3 and 100 mM KCl for the nitrate electrode. Immediately after back-filling, the electrode apices were frontfilled with commercially available ionophore cocktails for measuring hydrogen (#95297, Fluka) and ammonium (# 09879, Fluka). The nitrate sensor contained 0.5% (w/v) methyltridodecylammonium nitrate (MTDDA NO3 ), 0.084% (w/v) methyltriphenylphosphonium bromide (MTPPB) and 99.4% (v/v) n-phenyloctyl ether (NPOE) [11,12]. A reference electrode was fabricated in a similar way from a borosilicate glass capillary and filled with 0.1 M KCl in 2% (w/v) agar. Electrodes were calibrated against a range of standards (pH from 5.3 to 7.2, NH4+ and NO3 from 0.1 to 10 mM). Electrodes with responses less than 50 mV per decade were discarded. 2.3. Experimental procedures The roots were measured in Petri dishes placed on an inverted microscope. In the experiments to determine ammonium, nitrate and proton fluxes along the roots of aquatic plants, the bathing solution contained (in mM): NH4NO3 1.0; CaCl2 0.05; MgSO4 0.025; NaCl 0.56; K2SO4 0.025; NaH2PO4 0.032 and NaHCO3 0.2; pH 6.8–7.0. Thirty minutes after the pre-treatment with basal solution, measurements of ion fluxes commenced at 60–120 mm from the root surface. In our earlier studies we have shown that in media with NO3 , ion fluxes become stable 30 min after increase in NH4+ concentration [13]. In the mapping experiment, 3–4 replicate plants were measured and averaged. To cope with the oscillation pattern of ion fluxes in the elongation zone we measured meristem and elongation zones several times and the median value of ion fluxes for each plant was used for averaging between plants. Experiments were performed at 20–22 8C under standard laboratory lighting. Fluxes of H+, NH4+ and NO3 were measured at each root position simultaneously (see Fig. 2A–C). For estimation of plant preferences for NH4+ or NO3 supply, measurements were done on the same root position after 30 min of exposure to solution containing 1.0 mM of either NH4Cl, KNO3 or NH4NO3 in random order, pH 6.5–7.0. Each measurement took 5 mins (three root positions: apex and two points at the elongation zone). The Petri dish was then flushed with new solution containing the appropriate N source (NH4NO3 and NH4Cl). Flux measurements were performed 30 min after the transfer for plant adjustment to a new solution. Means and standard errors were calculated for 4–6 plants propagated and measured separately. Significant difference between means was assessed by Genstat 8th edition (VSN International Ltd., Hemel Hempstead, UK) for ANOVA analysis. 3. Results 3.1. H+ fluxes and pH profiles along the roots of aquatic plants Fluxes of H+, NO3 and NH4+ were measured at various positions (root apex, meristem, distal and proximal elongation
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Fig. 1. Root morphology under optical microscope. (A) Azolla spp.; (B) Bacopa monnieri; (C) Vallisneria natans; (D) Ludwigia repens.
zones) along the roots of four wetland plant species (Fig. 1A– D). Since Azolla spp. had a distinct root hair zone, fluxes were measured in that zone as well. The H+ fluxes demonstrated a highly specific pattern at different root zones (Fig. 2). The meristem zone was characterised as the zone with the highest H+ influx or the lowest efflux. In Azolla spp. and L. repens, the rate of H+ fluxes at the root apex was very low, around 0 nmol m 2 s 1. The apex and the meristem zone of V. natans roots were characterised by high H+ influx (around 60 nmol m 2 s 1). The distal and proximal elongation zone of Azolla spp., B. monnieri and L. repens showed H+ efflux in absolute values (Fig. 2). B. monnieri had the largest magnitude of H+ fluxes and the highest ability to
Table 1 Net fluxes (nmol m or NH4NO3) Species
2
acidify the basal media to a greater extent than other plant species tested (1.2 pH units in 2 h). 3.2. Mapping of NH4+ and NO3 fluxes along the roots of aquatic plants When NH4+ and NO3 were supplied together in B. monnieri, V. natans and L. repens, the highest net NH4+ uptake was measured in the meristem zone, decreasing at more mature regions (Fig. 2B). Specifically, the biggest difference between the high NH4+ uptake rate at the meristem zone and the low uptake rate at more mature regions was recorded for V. natans
s 1) of NH4+ and NO3 at the same points of the different root zones of four wetland species exposed to different media (1.0 mM NH4Cl, KNO3 Distance from tip (mm)
1.0 mM NH4Cl
1.0 mM KNO3
1.0 mM NH4NO3
NH4+
NO3 flux
NH4+ flux
flux
Azolla spp.
0 1200 2400
50 37ef 8 49e 106 19f
274 54cd 398 17c 296 61cd
Bacopa monnieri
0 1200 2400
81 42de 48 21e 47 28e
172 38d 255 49cd 273 86cd
Vallisneria natans
0 1200 2400
66 23ef 9 35e 3 37e
Ludwigia repens
0 1200 2400
88 18de 74 25de 68 55e
66 54def 23 43ef 38 32e
NO3 flux
NH4+ + NO3 flux
112 61f 47 71e 177 92d
178 76f 24 34e 215 65cd
250 53cd 108 47d 129 61de
14 23e 109 27d 196 29d
264 50cd 217 62cd 325 54c
74 25de 46 10e 74 26de
600 40 b 57 39e 18 39e
347 54f 37 75e 26 35e
253 64cd 20 68e 8 29e
30 28ef 8 43e 2 27e
372 56c 23 30e 74 40e
36 55e 665 43b 1015 53a
408 81bc 688 46b 1089 52a
Negative net values represent ion efflux, and positive values represent ion influx. Values are mean S.E.M. (n = 4–6). Different letters indicate significant differences ( p < 0.05) estimated by ANOVA.
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(from around 600 nmol m 2 s 1 down to zero), followed by L. repens (from around 400 nmol m 2 s 1 to zero). B. monnieri maintained the highest positive flux of NH4+ in the distal elongation zone among the species studied. Azolla spp.
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demonstrated the lowest variation in NH4+ spatial distribution along its root. In all four plant species studied, NO3 fluxes were the lowest at the root apex (Fig. 2C) and increased towards more mature
Fig. 2. Net H+ (A), NH4+ (B) and NO3 (C) fluxes along various root parts as noted in the top bars: root apex (hatched), meristem zone (black), distal elongation zone (white), proximal elongation zone (grey) and root hair zone (vertical lines). Flux measurement were taken while roots were exposed to the basal solution containing (in mM) NH4NO3 1.0; CaCl2 0.05; MgSO4 0.025; NaCl 0.56; K2SO4 0.025; NaH2PO4 0.032; and NaHCO3 0.2.
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Fig. 2. (Continued ).
regions peaking immediately behind the root apex. The relatively constant NO3 flux in the proximal elongation zone was the lowest in Valisneria natans (around 0 nmol m 2 s 1) and the highest in L. repens (around 1000 nmol m 2 s 1). 3.3. NH4+ and NO3 fluxes in relation to their availability A comparison was made between the rates of NH4+ and NO3 fluxes with different N source (NH4Cl, KNO3 or NH4NO3 ) in the bathing medium (Table 1). When both forms of N were available (1 mM NH4NO3 ), L. repens preferred NO3 for uptake at both points of the elongation zone (Table 1). All species except L. repens took up N effectively when NO3 was the sole source. Even when NH4+ was the only source of N (1 m NH4Cl), Azolla spp. did not switch on the NH4+ uptake system along the root segment monitored. A requirement for presence of both N forms in the bathing media was observed for L. repens; it accumulated NH4+ and NO3 in 1 mM NH4NO3, whereas it excluded NO3 in 1 mM KNO3 and NH4+ in 1.0 mM NH4Cl. When NH4+ and NO3 fluxes were summed in media containing1 mM NH4NO3 , L. repens had the largest N accumulation capacity, whereas Azolla spp. and V. repens took up N at a lower rate. 4. Discussion In this study, it was found that different aquatic plant species had different capacity to acidify external media: B. monnieri had the highest absolute values of H+ efflux (Fig. 2), showing a high capacity to acidify the media: it acidified and maintained
the pH of the bathing media at pH 5.4–5.6 (data not shown). This finding explains this species plasticity in regards to high external pH: B. monnieri usually is found in lakes with high pH [14]. The H+ fluxes in the media containing different forms of N were similar (data not shown), which is in accordance with studies on other plant species. In Lolium perenne and Trifolium repens, the H+ flux was generally unaffected by the concentration of external NO3 and NH4+ and did not correlate with the rates of N absorption [15]. Generally, the root apex, meristematic and apical elongation regions demonstrate net H+ influx in most plant species studied, with resulting alkalization of the adjacent medium [5]. Similarly, in our study all plants had the highest net H+ influx or the lowest H+ efflux at the root apex and meristem zones. However, the absolute values of H+ fluxes for three aquatic species, apart from V. natans, (Fig. 2) were relatively small compared with other studies [4,8,16]. Such small H+ flux could be explained by the high pH of our bathing solution (pH 6.5–7.0), compared to that in other studies (pH 5.5–6.0). However, this pH is closer to that of native ecosystems (pH 7–8; Fang, unpublished). As expected, there was a consistent relationship between the fluxes of NH4+ and H+ measured simultaneously at each root position for all plants studied: Azolla spp. (r = 0.57); B. monnieri (r = 0.73); V. natans (r = 0.95); and L. repens (r = 0.85). Higher NH4+ uptake rate coincided with higher H+ uptake and increased pH in the root apex zone (data not shown). Therefore, our results provide additional information to a hypothesis that NH4+ toxicity is explained by acidification of the external media during NH4+ supply as the sole N source,
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showing that the observed in other studies [17] acidification is not directly linked to H+ extrusion during NH4+ uptake. The high correlation between NH4+ and H+ fluxes are in agreement with studies performed on eucalypt roots where authors used the same MIFE technique [4], which calculates ion fluxes more comprehensively, analysing diffusion rate of ions at the particular area of the root [5]. However, in the majority of other studies H+ flux was estimated as a change of the bulk pH, which could reflect different reasons including a change in the concentration of other ions (e.g. carbonate). Also, kinetics analysis of NH4+ uptake, estimated by measurements of plasma membrane potential, allowed authors to suggest H+–NH4+ symport in rice roots [18]. In addition to different techniques used for studying the interdependence of NH4+ and H+ transport, other factors could be responsible for the contradicting results, such as the time of plant exposure to different N sources or the different experimental setup (‘‘disturbed’’ or ‘‘undisturbed’’ conditions). From our study, it is plausible to suggest that different root zones prefer different forms of N, when both forms of N are available in the surrounding media. A larger net influx of NH4+ was observed in the meristem zone in comparison with the elongation region (Fig. 2B, Table 1). Similar results were observed for rice when plants were exposed to NH4+ and NO3 simultaneously [8]. For maize roots these researchers also observed a small decline in net NH4+ uptake in the elongation zone compared with the root apex and meristem zone. Nitrate uptake was higher at the elongation zone than in other root regions in all four species studied (Fig. 2C, Table 1). Similarly, Siebrecht et al. [19] found that NO3 uptake into the apex of barley roots was only half of that into the older root zones. When both NH4+ and NO3 were supplied together, the root apices exhibited higher net influx (or smaller net efflux) of NH4+ than NO3 , but the magnitude differed among species (Table 1). Net NH4+ uptake was 15-fold greater than net NO3 uptake in B. monnieri and 10-fold greater in L. repens. Similarly, net NH4+ uptake was 2-fold greater than net NO3 uptake in the maize root apex zone [8], and 3-fold greater in rice roots [7]. Several explanations can be suggested for the observed behaviour in this clearly defined root zone, where plants are taking NH4+ at a higher rate than NO3 . The first explanation suggested based on root morphology: different root tissues demand different amounts of NH4+ and NO3 and the meristem zone requires a higher amount of NH4+ for protein synthesis [8]. The second explanation is based on the different expression and activities of transport systems for both NH4+ and NO3 in different root zones. Net NH4+ and NO3 uptake can be mediated by high-affinity transporters as well as various low-affinity transporters, and can be reversed by NH4+ and NO3 efflux systems. To date, several transporters have been cloned: high affinity NH4+ transporters from the ammonium transporters (AMT) family and NO3 transporters from nitrate– nitrite (NNP) and peptide transporters (PTR) [20,21]. However, there is no information on their expression pattern along the root apex. No specific transporters that function as NH4+ or NO3 efflux transporters have been cloned yet.
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Our study demonstrated again that different plant species require different forms of N [21]. From four plants studied, two (B. monnieri and Azolla spp.) had preference for NO3 , whereas both N forms were required by L. repens for N uptake (Table 1). The limited ability of Azolla spp. plants to take up NH4+, which was observed in our study as NH4+ efflux in mono-NH4+ or mixed (NH4+ and NO3 ) media, is consistent with their biological requirements. Being a symbiont with Anabaena azollae, a nitrogen fixing cyanobacteria, Azolla plants are usually well supplied with NH4+ [22]. Plant preference for different forms of N is influenced by environmental factors such as root- or air-temperature, aeration, solution pH, composition of the culture solution, water stress and high concentration of salts in the root zone, and also by the plant growth stage and its ability to form a symbiosis with bacteria or fungi [22–24]. It could be suggested that these different N-uptake requirements depend on different growth rates or phenological phases of the plants studied. The precise mechanisms underpinning NH4+/NO3 interactions in aquatic species should be further investigated. Therefore, for industrial purposes, when plants are used for removing N from eutrophicated water, plants species should be selected according to their preferences for different N forms.
Acknowledgements We are grateful to Dr. Ian Newman, the University of Tasmania, who kindly lent us the MIFE system for selective ion measurements. Root pictures were taken in the Centre for Microscopy and Microanalysis, the University of Western Australia. The project was supported by the China Fund (administered by the Australian Department of Education, Science and Training).
References [1] T. Gumbricht, Nutrient removal processes in freshwater submersed macrophyte systems, Ecol. Eng. 2 (1993) 1–30. [2] Y.X. Chong, H.Y. Hu, Y. Qian, Advances in utilization of macrophytes in water pollution control, Tech. Equip. Env. Pol. Contr. 4 (2004) 36–40, in Chinese. [3] F. Wu, H. Qing, G. Wan, Regeneration of N, P and Si near the sediment/ water interface of lakes from Southwestern China Plateau, Water Res. 35 (2001) 1334–1337. [4] T.P. Garnett, S.N. Shabala, P.J. Smethurst, I.A. Newman, Simultaneous measurement of ammonium, nitrate and proton fluxes along the length of eucalypt roots, Plant Soil 236 (2001) 55–62. [5] I.A. Newman, Ion transport in roots, measurement of fluxes using ionselective microelectrodes to characterize transporter function, Plant Cell Environ. 24 (2001) 1–14. [6] G.H. Henriksen, D.R. Raman, L.P. Walker, R.M. Spanswick, Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes. II. Patterns of uptake along the root axis and evaluation of the microelectrode flux estimation technique, Plant Physiol. 99 (1992) 734–747. [7] T.D. Colmer, A.J. Bloom, A comparison of NO3 and NH4+ net fluxes along roots of rice and maize, Plant Cell Environ. 21 (1998) 240–246. [8] A.R. Taylor, A.J. Bloom, Ammonium, nitrate, and proton fluxes along the maize root, Plant Cell Environ. 21 (1998) 1255–1263.
246
Y.Y. Fang et al. / Plant Science 173 (2007) 240–246
[9] Y.Y. Fang, O. Babourina, Z. Rengel, X.E. Yang, P.M. Pu, Ammonium and nitrate uptake by the floating plant Landoltia punctata, Ann. Bot. 99 (2007) 365–370. [10] J. Gerenda´s, Z. Zhu, R. Bendixen, R.G. Ratcliffe, B. Sattelmacher, Physiological and biochemical processes related to ammonium toxicity in higher plants, Z. Pflanzen. Boden. 160 (1997) 239–251. [11] R.G. Zhen, S.J. Smith, A.J. Miller, A comparison of nitrate-selective microelectrodes made with different nitrate sensors and the measurement of intracellular nitrate activities in cells of excised barley roots, J. Exp. Bot. 43 (1992) 131–138. [12] C. Plassard, A. Gue´rin-Laguette, A.A. Ve´ry, V. Casarin, J.B. Thibaud, Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effect of ectomycorrhizal symbiosis, Plant Cell Environ. 25 (2002) 75–84. [13] O. Babourina, K. Voltchanskii, B. McGann, I. Newman, Z. Rengel, Nitrate supply affects ammonium transport in canola roots, J. Exp. Bot. (2007) 651–658. [14] M.V. Hoyer, D.E. Canfield, C.A. Horsbourgh, K. Brown, Florida Freshwater Plants: A Handbook of Common Aquatic Plants in Florida Lakes, University of Florida, Institute of Food and Agricultural Sciences, Gainesville, FL, 1996. [15] H. Høgh-Jensen, B. Wollenweber, J.K. Schjoerring, Kinetics of nitrate and ammonium absorption and accompanying H+ fluxes in roots of Lolium perenne L. and N2-fixing Trifolium repens L., Plant Cell Environ. 20 (1997) 1184–1192.
[16] T.P. Garnett, S.N. Shabala, P.J. Smethurst, I.A. Newman, Kinetics of ammonium and nitrate uptake by eucalypt roots and associated proton fluxes measured using ion selective microelectrodes, Funct. Plant Biol. 30 (2003) 1165–1176. [17] A.L. Miller, G.N. Smith, J.A. Raven, N.A. Gow, Ion currents and the nitrogen status of roots of Hordeum vulgare and non-nodulated Trifolium repens, Plant Cell Environ. 14 (1991) 559–567. [18] M.Y. Wang, A.D.M. Glass, J.E. Shaff, L.V. Kochian, Ammonium uptake by rice roots. III. Electrophysiology, Plant Physiol. 104 (1993) 899–906. [19] S. Siebrecht, G. Ma¨ck, R. Tischner, Function and contribution of the root apex in the induction of NO3 uptake along the barley root axis, J. Exp. Bot. 46 (1995) 1669–1676. [20] B.G. Forde, Nitrate transporters in plants: structure, function and regulation, Biochem. Biophys. Acta 1465 (2000) 219–235. [21] D. Loque, N. von Wiren, Regulatory levels for the transport of ammonium in plant root, J. Exp. Bot. 55 (2004) 1293–1305. [22] J.C. Meeks, N.A. Steinberg, C.S. Enderlin, C.M. Joseph, G.A. Peters, Azolla–Anabaena relationship. XIII. Fixation of [13N]N2, Plant Physiol. 84 (1987) 883–886. [23] H. Ikeda, Utilization of nitrogen by vegetable crops, Jap. Agricult. Res. Quart. 25 (1991) 117–124. [24] T.P. Garnett, P.J. Smethurst, Ammonium and nitrate uptake by Eucalyptus nitens, effect of pH and temperature, Plant Soil 214 (1999) 133–140.