Martins-Loução MA, Cruz C. Plant 15∂N as an indicator of NH4+ tolerance. In "Nutrición Mineral, aspectos fisiológicos, agronómicos y ambientales”. Eds. Carmen Lamsfus Arrien (editora-coordinadora), Pedro M. Aparicio Tejo, César Arrese-Igor Sánchez, Ignacio Irigoyen Iriarte, José Fernando Morán Juez, Universidad Pública de Navarra. Pp 157-164.

Plant ∂ 15N as an indicator of NH4+ tolerance Cristina Cruz1, María D. Domínguez-Valdivia2,José Morán2, Carmen Lamsfus2, Pedro M. Aparicio-Tejo2, Maria Amélia Martins-Loução1 (1) Universidade de Lisboa, Faculdade de Ciências, Centro de Ecologia e Biologia Vegetal Campo Grande. C2. Piso 4. 1749-016 Lisboa (2) Dpto. Ciencias del Medio Natural Universidad Pública de Navarra. Campus Arrosadía s/n 31006 Pamplona E-mail: [email protected] Introduction Sensitivity to (ammonium) NH4+ may be an universal biological phenomenon as it has been observed in almost all living systems (Britto and Kronzucker 2002). However, the threshold at which symptoms become manifested among plant species differs widely. Although contradictory results have been obtained, mainly due to varying experimental conditions, it is possible to generalize that crops are more sensitive to NH4+ toxicity than wild species, especially those characteristic of the latter stages of the natural succession (Martins-Loução and Cruz 1999; Britto and Kronzucker 2002). The causes of NH4+ toxicity are not clear: in over 80 years, numerous studies about this subject have not arrived at a definitive conclusion. Plant growth in response to NH4+ can be considered as a function of several effects: NH4+ induced mineral nutrition deficiency caused by impaired uptake of cations, acidification of the rhyzosphere (Lee and Ratcliffe 1991), alterations in the osmotic balance or modified hormone metabolism (Claussen and Lenz 1999; MartinsLoução and Cruz 1999; Britto and Kronzucker 2002). Conditions supporting a rapid metabolization of NH4+N in the roots such as a pH above a certain threshold level in the rhyzosphere and sufficient carbohydrate reserves in the roots contribute to prevent transport of NH4+ to the shoot (Schjoerring et al 2002), which is considered to be more sensitive to NH4+ accumulation than the root. Although mechanisms underlying NH4+ toxicity have been extensively sought the causes conferring it at the cellular level are not understood. In this work we used the ∂15N as an integrator of the N metabolism at the plant level in order to highlight possible causes for NH4+ toxicity.

Materials and Methods Seeds of spinach (Spinacia oleracea L. cv Spinner), tomato (Lycopersicon esculentum L. cv Trust), lettuce (Lactuca sativa L. cv Marine), pea (Pisum sativum L. cv Eclipse) all from Johnny’s Selected seeds, Maine, USA and lupinus (Lupinus albus L. cv albus) from Cebeco Seed Innovations LTD, Norfolk, UK, were sawn in absorbent paper and allowed to germinate and grow for one week. Humidity was maintained relatively constant by periodically adding distilled water. Seedlings in good condition and in similar stage of development (number of leaves) were transferred to pots containing distilled water. Pots (2 l capacity), with 3 plants each, were kept in a growth chamber with a photoperiod of 12 h (from 8:00 to 20:00), air temperature of 24/20˚C (day/night), air relative humidity of 69–73%, and light intensity of 550 µmol m-2s-1 photosynthetic photon flux, obtained from 12 lamps Philips TLD 18W/84, UK and 4 Sylvania GRO-Lux IF 18W+ GRO, Germany. After 2 days in distilled water, plants of each species were divided in two groups, each receiving 2 different concentration of N, either in the form of NO3- or NH4+. Each group of plants consisted in 6 pots (18 plants). Plants were grown for three weeks in hydroponic culture. Modified Hoagland solutions, 1/4 strength, were used in order to obtain several concentrations (1.5 and 3.0) of NO3- and NH4+. All plants received 0.5 mM MgPO4, 50 µM KCl, 25 µM H3BO3, 2 µM MnSO4.H2O, 2 µM ZnSO4.7H2O, 0.5 µM CuSO4.5H2O, 0.5 µM (NH4)6Mo7O24, and 20 µM FeNaEDTA. NO3- was applied as mixture of potassium and calcium NO3-, and NH4+ as (NH4)2SO4. Potassium concentrations in

the solutions were kept constant by addition of potassium sulphate. Temperature of the solutions was kept between 18 and 20˚C. Solutions were always checked for their NO3- and NH4+ concentrations before and after being in contact with the plants (results not shown). Nutrient solutions were vigorously aerated (flow rate of 15 ml s-1), and changed every two days. Seedlings were allowed to grow under these conditions for 3 weeks before collection. Plants were divided in roots and shoots and each part was weighted. Samples were collected for dry weight determination, after drying the material at 60˚C until constant weight (48 h). From each pot, 5-8 mg of powered plant material (leaves and roots were taken separately) was packed in tin capsules and analyzed by isotope ratio mass spectrometry for the 15N/14N isotope ratios (Isoprime isotope ratio mass spectrometer – IRMS, Micromass-GV Instruments, UK). The results of the carbon isotope composition are expressed as ∂15N in parts per thousand (‰) relative to the atmospheric N2: ∂15N (‰) = (Rsample/Rstandard-1)*1000. Where Rsample is the 15 N/14N ratio of the sample and Rstandard is the 15N/14N ratio of atmospheric N2. For batch calibration in the isotope ratio analyses plant material, previously calibrated against standard material of known isotope composition, were used as working standard. Results and Discussion Our results show that, in terms of biomass production, plant responses varied between species, depending on either or both N source and concentration (Table. 1). Lupinus and pea presented identical biomass accumulation in the presence of NO3- or NH4+. Their biomass production was hence apparently independent of the N source in the medium but very much influenced by N concentration (increasing either NO3- or NH4+ concentration lead to higher biomass accumulation). Therefore they were considered as NH4+tolerant. There were other species, like lettuce, for which the N source, NO3- or NH4+, strongly affected biomass accumulation. NH4+concentrations allowing maximal biomass accumulation vary considerably between species, and tend to be lower than those of NO3- for NH4+ sensitive species (Cruz et al 2006). Table 1 Biomass accumulation (g plant -1) by plants of 5 species: lettuce (Lactuca sativa L.) spinach (Spinacia oleracea L.), tomato (Lycopersicon esculentum L.), pea (Pisum sativum L.) and Lupinus (Lupinus albus L.) grown for 3 weeks in distinct concentrations (1.5 and 3.0 mM) of NH4+ and NO3-. Symbols represent mean values ± SD (n = 10). Treatment Plant species (mM) Lettuce Spinach Tomato Pea Lupinus 1.5 NH4+ 0.4 ± 0.01 1.4 ± 0.11 2.5 ± 0.19 1.02 ± 0.09 1.7 ± 0.12 3.0 NH4+ 0.6 ± 0.12 1.1 ± 0.12 1.1 ± 0.12 1.21 ± 0.20 2.3 ± 0.11 1.5 NO3 0.8 ± 0.02 1.6 ± 1.1 2.5 ± 0.14 0.8 ± 0.02 1.8 ± 0.21 3.0 NO31.0 ± 0.01 2.3 ± 0.31 3.0 ± 0.21 1.0 ± 0.10 2.3 ± 0.22 The results obtained for the ∂15N of the whole plants clearly showed that: (1) plants grown with NO3- as the only N source were enriched and plants grown with NH4+ as the only N source were impoverished in 15N relatively to the respective N source; and (2) higher tolerance to NH4+, assessed by biomass accumulation with NO3- and NH4+ at equivalent concentrations, is related with less impoverishment in 15N of the whole plant (Table 2). Therefore the first conclusion to be taken is that N source has strong implications on plant metabolism. Discrepancies between the whole plant ∂ 15N and that of the N sources reflect the existence of discriminative processes in influxes and/or effluxes between the plant and the environment (Hogberg 1997). The reported values for ∂15N during the uptake of NO3- are contradictory and apparently dependent on NO3- concentration, type of substrate, plant species, plant age, etc (Mariotti et al 1982, Evens et al 1996). Independent of the mechanism, and the individual steps, the proper uptake of NO3- by higher plants does proceed without notable N isotope discrimination (Yoneyama et al 2001); the in vivo measured ∂15N is hence probably mainly caused by N isotope effect on the assimilatory NO3- metabolization. However, the

discrimination, if present, would contribute to explain an impoverishment of the plant material in 15N and not an enrichment as was observed (Table 2). The enrichment of the nit NO3--fed plant material in 15N can be explained if: the influx is not comparatively discriminative in relation to the efflux; and/or the N pool that is subjected to efflux is itself more enriched than the external N pool (Robinson 2001). This interpretation would also explain the enrichment in 15 N of the legumes (pea and lupinus) in relation to the other plant species (lettuce, spinach and tomato). In pea and lupinus roots are the main place for N metabolism, therefore most of the N present in the root exudates is in the organic form. As most of the enzymes discriminate against the heaviest isotope, loosing organic N implies getting enriched relatively to the medium (Werner and Schmidt 2004). Table 2 ∂15N of leaves and roots of 5 plant species: lettuce (Lactuca sativa L.) spinach (Spinacia oleracea L.), tomato (Lycopersicon esculentum L.), pea (Pisum sativum L.) and Lupinus (Lupinus albus L.) grown for 3 weeks in distinct concentrations (1.5 and 3.0 mM) of NH4+ and NO3-. Symbols represent mean values ± SD (n = 10). Source ∂15N: NO3- = 0.3 and NH4+ = 0.5 Treatment Plant species (mM) Lettuce Spinach Tomato Pea Lupinus -5.12 ± 0.23 -3.45 ± 0.31 -0.21 ± 0.01 -0.30 ± 0.22 1.5 NH4± Leaf -7.38 ± 0.12 -9.77 ± 1.00 -6.54 ± 0.45 0.24 ± 0.12 0.03 ± 0.12 Root -12.05 ± 2.3 ± -7.20 ± 1.2 -5.62 ± 0.32 -5.45 ± 0.16 -3.89 ± 0.41 -3.12 ± 0.51 3.0 NH4 Leaf -8.12 ± 055 -6.12 ± 0.23 -3.45 ± 0.23 -3.32 ± 0.21 Root -14.54 ± 3.2 5.02 ± 0.8 4.93 ± 0.12 7.32 ± 1.21 10.65 ± 0.21 12.9 ± 0.21 1.5 NO3Leaf 4.81± 0.11 3.26 ± 0.23 7.51 ± 1.32 9.23 ± 2.22 8.12 ± 1.11 Root 3.12 ± 012 4.89 ± 0.33 8.01 ± 1.12 11.77 ± 2.1 13.1 ± 1.1 3.0 NO3 Leaf 2.99 ± 0.12 3.55 ± 0.21 6.54 ± 0.24 10.00 ± 2.1 11.24 ± 1.2 Root As far as NH4+is concerned the whole plant is impoverished in 15N in relation to the external source of NH4+, mainly in NH4+susceptible species (Table 2). This implies the existence of a strong discrimination in the uptake step of NH4+acquisition. Variations in stable isotope ratios are the result of equilibrium and kinetic isotope effects. A larger activation energy is required to dissociate an isotopically heavy chemical species than a light one. Hence, an isotopically light atom or ion will be bonded less strongly at equilibrium, in the case of NH4+/ NH3 , NH4+ is more enriched (20‰) with 15N than NH3 (Hogberg 1997). Accordingly the larger the impoverishment in 15N, the larger the contribution of an uptake system favouring NH3 in relation to NH4+. Low and high affinity transport systems have been described for NH4+uptake by plants. Their regulation is both at the translational and post-translational level. However, both systems transport charged species (NH4+), with low or none effect on the N isotope fractionation, since in aqueous system at pH 7.6 more than 99% of the NH4+will be in the protonated form. However, a family of membrane proteins transporting NH3 (and not NH4+) has been identified and described (Khademi et al, 2004). The structural analysis of this protein (AmtB) is based on a recruitment vestibule for cations such as NH4+ or neutral NH3, a site that can bind NH4+ using π-cation interactions, and an hydrophobic channel that incorporates NH3 using weak interactions with C-H hydrogen bond donors. The H+ stays outside the membrane and the NH3 reaches the cytoplasm of the cell. This mechanism implies the dissociation of NH4+, which will occur predominantly in the lighter forms of ion, allowing for discrimination in the uptake. Yoneyama et al (2001) suggested that NH4+ transport mechanisms of plants growing poorly with NH4+nutrition should be investigated, and suggest that poor development of the roots of such plants may reflect the dominance of the diffusion mediated mechanism in relation to the carriermediated active transport of NH4+. According to our results, the AmtB transport mechanism would be relatively more important in species like lettuce and spinach, less tolerant to NH4+ (Tables 1 and 2). The connection between low biomass accumulation, 15N impoverishment, and relative importance of AmtB in NH4+ transport may rely on its implications to the pH of the root

cell. At cytosolic pH the NH3 entering the cell will be immediately protonated (pKa of NH4+ = 9.25) leading to cytosolic alkalinization (Britto and Kronzucker, 2005). The proton may be recovered if NH4+ will be metabolised, but not if NH4+ will be stored, exported to the shoot or effluxed. Increased N metabolization rates, e.g. high GS activities, are characteristic of plant response to NH4+. Lettuce, spinach and tomato accumulate relatively high concentrations of NH4+ in leaves (Cruz et al, 2006). NH4+ volatilization from leaves is a mechanism for NH4+ detoxification. NH3 volatilization involves several steps in which isotopic fractionation can occur: the equilibrium of NH4+/NH3 in solution, diffusion of NH3 to the site of volatilization, volatilization of NH3 and its diffusion from the site of volatilization. The compounded effect of these processes on the net fractionation can be large (-40‰ relatively to the substrate, Handley et al, 1997) and may justify the less negative ∂15N of the leaves in relation to the roots of NH4+ fed plants (Table 2). References Britto DT and Kronzucker HJ 2002 NH4+ toxicity in higher plants: a critical review. Journal of Plant Physiology 159: 567–584. Britto DT and Kronzucker HJ 2005 N acquisition, PEP carboxylase, and cellular pH homeostasis: new views on old paradigms. Plant, Cell and environment 28, 1396-1409. Claussen W, Lenz F 1999 Effect of NH4+or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant and Soil 208: 95–102 Cruz C, Bio AMF, Domínguez-Valdivia MD, Aparicio-Tejo PM, Lamsfus C and MartinsLoução MA 2006 How does glutamine synthetase activity determine plant tolerance to NH4+? Planta 223, 1068–1080 Evans RD, Bloom AL, Sukrapanna SS and Ehleringer JR 1996 N isotop composition of tomato (Lycopersicum esculentum Mill. cv. T-5) grown under NH4+ or nitrate nutrition. Plant, Cell and Environment 19: 1317-1323. Handley LL, Robinson D, Forster BP, Ellis RP, Scrimgeour CM, Gordon DC, Nevo E and Raven JA 1997 Shoot 15N correlates with genotype and salt stress in barley. Planta, 201 100102. Högberg P 1997 15N natural abundance in soil-plant systems. New Phytologist 137, 179-203. Khademi S, O’Connell III J, Remis J, Robles-Colmenares Y, Miercke LJW and Stoud RM 2004 Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305, 1587-1594. Lee RB, Ratcliffe RG 1991 Observations on the subcellular distribution of the NH4+ ion in maize root tissue using in vivo 14N-nuclear magnetic resonance spectroscopy. Planta 183: 359367. Mariotti A, Mariotti F, Champigny M-L, Amargar N and Moyse A 1982 N isotop fractionation associated with nitrate reductase activity and uptake of nitrate by pearl millet. Plant Physiology 69, 880-884. Martins-Loução MA and Cruz C 1999 Role of N source in carbon balance. In: Srivastava (ed.) N Nutrition and Plant Growth. New Delhi, pp 231–282 Robinson D 2001 Delta 15N as an integrator of the N cycle. Plant and Soil 16, 153-162. Schjoerring JK, Husted S, Mack G and Mattsson M 2002 The regulation of NH4+ trasnlocation in plants. J Exp Bot 53: 883–890. Werner RL and Schmidt H-L 2004 The in vivo N isotope discrimination among organic plant compounds. Phytochemistry 61, 465-484 YoneyamaT, Matsumaru T, Usut K and Engellar WMHG 2001 Discrimination of N isotopes during absorption of NH4+ and nitrate at different N concentrations by rice (Oryza sativa L.) plants. Plant, Cell and environment 24, 133-139.