Isolation and characterization of a high-affinity ammonium transporter ApAMT1;1 in alligatorweed

  • Xiaotong Guo
  • Yuting Sheng
  • Shunying Yang
  • Lei Han
  • Yachao Gao
  • Kai Zhang
  • Jieshan Cheng
  • Hongxia Zhang
  • Zhizhong SongEmail author
  • Yanhua SuEmail author
Original paper


In aquatic fields, ammonium (NH4+) is the most preferred nitrogen (N) source used by plants. The uptake of NH4+ is facilitated by the family of ammonium transporters (AMTs). However, the molecular functions of AMTs in aquatic plants are largely unknown. In this work, a new NH4+ transporter encoding gene, ApAMT1;1, was isolated from the typical aquatic plant alligatorweed, using degenerated primers and rapid amplification of cDNA end (RACE) techniques. Quantitative real time PCR showed that ApAMT1;1 was predominantly expressed in roots, and significantly induced by NH4+ starvation in all tested tissues, including leaves, stems and roots. Functional determination and 15N-labeled ammonium uptake assays in yeast cells indicated that ApAMT1;1 was a typical high-affinity transporter, with a 38.6 μM Km value, and the phosphorylation site T469 was required to retain its NH4+ uptake capacity. Further analyses with Met sulfoximine (MSX), a NH4+ assimilation inhibitor, demonstrated that ApAMT1;1-mediated NH4+ uptake might be feedback regulated by the internal NH4+ accumulation. Our results reveal a functional role of ApAMT1;1 in the uptake and transport of NH4+ in aquatic plants.


Alligatorweed Ammonium transporter ApAMT1;1 Aquatic plant NH4+ uptake 



We are grateful for Dr. Julia Davies and Dr. Elsa Matthus (Department of Plant Science, University of Cambridge) for critical comments on this work and sincere help during the studies in University of Cambridge.


This work was financially supported by grants from the National Natural Science Foundations of China (Grant Nos. 31601819, 31700524 and 31801837), Doctoral Fund of Shandong Natural Science Foundation (Grant Nos. ZR2016CB19 and ZR2016CB48), Science and Technology Project of Yantai (Grant No. 2016ZH058), Key R&D Program of Shandong Province (Grant No. 2017NC210012), and The Modern Agricultural Industry Technology System Innovation Team of Shandong Province of China (Grant No. SDAIT-02-05).

Compliance with ethical standards

Conflict of interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Supplementary material

10725_2019_537_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 33 kb)
10725_2019_537_MOESM2_ESM.doc (38 kb)
Supplementary material 2 (DOC 38 kb)
10725_2019_537_MOESM3_ESM.tif (1.1 mb)
Supplementary material 3 Amino acid sequence alignment of AMT1 homologs. Alignment was carried out by the bootstrap option of the CLUSTAL W multiple alignment packages. GenBank accession numbers of these AMT1 members used in the tree establishment are listed in Supplemental Table 2. (TIFF 1112 kb)


  1. Bai QF, Zheng BH, Tian ZQ (2004) Ecological effects of aquatic plants on water pollution control. Environ Sci Technol 27:99–110 (in Chinese) Google Scholar
  2. Betteridge PR, Ayling P (1975) The role of methionine transport defective mutations in resistance to methionine sulphoximine in Salmonella typhimurium. Mol Gene Genet 138:41–52CrossRefGoogle Scholar
  3. Britto DT, Kronzucker HJ (2002) NH4 + toxicity in higher plants: a critical review. J Plant Physiol 159:567–584CrossRefGoogle Scholar
  4. Britto DT, Siddiqi MY, Glass AMD, Kronzucker HJ (2001) Futile transmembrane NH4 + cycling: a cellular hypothesis to explain ammonium toxicity in plants. Proc Natl Acad Sci USA 98:4255–4258CrossRefGoogle Scholar
  5. Cerezo M, Tillard P, Gojon A, Primo-Millo E, Garcia-Agustin P (2001) Characterization and regulation of ammonium transport systems in Citrus plants. Planta 214:97–105CrossRefGoogle Scholar
  6. Chiasson DM, Loughlin PC, Mazurkiewicz D, Mohammadidehcheshmeh M, Fedorova EE, Okamoto M, McLean E, Glass AD, Smith SE, Bisseling T, Tyerman SD, Day DA, Kaiser BN (2014) Soybean SAT1 (symbiotic ammonium transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4 + transport. Proc Natl Acad Sci USA 11(13):4814–4819CrossRefGoogle Scholar
  7. Couturier J, Montanini B, Martin F, Brun A, Blaudez D, Chalot M (2007) The expanded family of ammonium transporters in the perennial poplar plant. New Phytol 174:137–150CrossRefGoogle Scholar
  8. Cruz C, Bio AFM, Dominguez-Valdivia MD, Aparicio-Tejo PM, Lamsfus C, Martins-Loucao MA (2006) How does glutamine synthetase activity determine plant tolerance to ammonium? Planta 223:1068–1080CrossRefGoogle Scholar
  9. D’Apuzzo E, Rogato A, Simon-Rosin U, El Alaoui H, Barbulova A, Betti M, Dimou M, Katinakis P, Marquez A, Marini AM, Udvardi MK, Chiurazzi M (2004) Characterization of three functional high-affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression. Plant Physiol 134:1763–1774CrossRefGoogle Scholar
  10. Dortch Q (1990) The interaction between ammonium and nitrate uptake in phytoplankton. Mar Ecol-Prog Ser 61:183–202CrossRefGoogle Scholar
  11. Gazzarrini S, Lejay L, Gojon A, Ninnemann O, Frommer WB (1999) Three functional transporters for constitutive, diurnally regulated and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11:937–947CrossRefGoogle Scholar
  12. Giehl RFH, Laginha AM, Duan F, Rentsch D, Yuan L, von Wirén N (2017) A critical role of AMT2;1 in root-to-shoot translocation of ammonium in Arabidopsis. Mol Plant 10(11):1449–1460CrossRefGoogle Scholar
  13. Graff L, Obrdlik P, Yuan L, Loqué D, Frommer WB, von Wirén N (2011) N-terminal cysteines affect oligomer stability of the allosterically regulated ammonium transporter LeAMT1;1. J Exp Bot 62:1361–1373CrossRefGoogle Scholar
  14. Guo H, Wang N, McDonald TR, Reinders A, Ward JM (2018) MpAMT1;2 From Marchantia polymorpha is a high-affinity, plasma membrane ammonium transporter. Plant Cell Physiol 59(5):997–1005CrossRefGoogle Scholar
  15. Hao D, Yang S, Huang Y, Su Y (2016) Identification of structural elements involved in fine-tuning of the transport activity of the rice ammonium transporter OsAMT1;3. Plant Physiol Biochem 108:99–108CrossRefGoogle Scholar
  16. Hildebrand M (2005) Cloning and functional characterization of ammonium transporters from the marine diatom Cylindrotheca fusiformis (Bacillariophyceae). J Phycol 41:105–113CrossRefGoogle Scholar
  17. Kakinuma M, Nakamoto C, Kishi K, Coury DA, Amano H (2017) Isolation and functional characterization of an ammonium transporter gene, PyAMT1, related to nitrogen assimilation in the marine macroalga Pyropia yezoensis (Rhodophyta). Mar Environ Res 128:76–87CrossRefGoogle Scholar
  18. Kronzucker HJ, Siddiqi MY, Glass ADM (1997) Root discrimination against soil nitrate and the ecology of forest succession. Nature 385:59–61CrossRefGoogle Scholar
  19. Lanquar V, Loqué D, Hörmann F, Yuan LX, Bohner A, Engelsbergerd WR, Lalonde S, Schulze WX, von Wirén N, Frommer WB (2009) Feedback inhibition of ammonium uptake by a phospho-dependent allosteric mechanism in Arabidopsis. Plant Cell 21:3610–3622CrossRefGoogle Scholar
  20. Lauter FR, Ninnemann O, Bucher M, Riesmeier JW, Frommer WB (1996) Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato. Proc Natl Acad Sci USA 93:8139–8144CrossRefGoogle Scholar
  21. Li T, Liao K, Xu X, Gao Y, Wang Z, Zhu X, Jia B, Xuan Y (2017) Wheat ammonium transporter (AMT) gene family: diversity and possible role in host-pathogen interaction with stem rust. Front Plant Sci 8:1637CrossRefGoogle Scholar
  22. Liu Y, von Wirén N (2017) Ammonium as a signal for physiological and morphological responses in plants. J Exp Bot 68(10):2581–2592CrossRefGoogle Scholar
  23. Liu Y, Sun J, Tian Z, Hakeem A, Wang F, Jiang D, Cao W, Adkins SW, Dai T (2017) Physiological responses of wheat (Triticum aestivum L.) germination to elevated ammonium concentrations: reserve mobilization, sugar utilization, and antioxidant metabolism. Plant Growth Regul 81:209–220CrossRefGoogle Scholar
  24. Loqué D, von Wirén N (2004) Regulatory levels for the transport of ammonium in plant roots. J Exp Bot 55:1293–1305CrossRefGoogle Scholar
  25. Loqué D, Lalonde S, Looger LL, von Wirén N, Frommer WB (2007) A cytosolic trans-activation domain essential for ammonium uptake. Nature 446:195–198CrossRefGoogle Scholar
  26. Loqué D, Mora SI, Andrade SLA, Pantoja O, Frommer WB (2009) Pore mutations in ammonium transporter AMT1 with increased electrogenic ammonium transport activity. J Biol Chem 284:24988–24995CrossRefGoogle Scholar
  27. Ludewig U, von Wirén N, Frommer WB (2002) Uniport of NH4 + by the root hair plasma membrane ammonium transporter LeAMT1;1. J Biol Chem 277:13548–13555CrossRefGoogle Scholar
  28. Ludewig U, Neuhäuser B, Dynowski M (2007) Molecular mechanisms of ammonium transporter and accumulation in plants. FEBS Lett 581:2301–2308CrossRefGoogle Scholar
  29. Marini AM, Soussi-Boudekou S, Vissers S, Andre B (1997) A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17:4282–4293CrossRefGoogle Scholar
  30. Mayer M, Schaaf G, Mouro I, Lopez C, Colin Y, Neumann P, Cartron JP, Ludewig U (2006) Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters. J Gen Physiol 127:133–144CrossRefGoogle Scholar
  31. McDonald TR, Ward JM (2016) Evolution of electrogenic ammonium transporters (AMTs). Front Plant Sci 7:352CrossRefGoogle Scholar
  32. Murashige T, Skoog F (1962) A revised medium for the rapid growth and bioassays with tobacco cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  33. Neuhäuser B, Dynowski M, Mayer M, Ludewig U (2007) Regulation of NH4 + transport by essential cross talk between AMT monomers through the carboxyl tails. Plant Physiol 143:1651–1659CrossRefGoogle Scholar
  34. Neuhäuser B, Ludewig U (2014) Uncoupling of ionic currents from substrate transport in the plant ammonium transporter AMT1;2. J Biol Chem 289(17):11650–11655CrossRefGoogle Scholar
  35. Ninnemann O, Jauniaux JC, Frommer WB (1994) Identification of a high affinity NH4 + transporter from plants. EMBO J 13:3464–3471CrossRefGoogle Scholar
  36. Ramarkers C, Ruijter JM, Lekanne Deprez RH, Moorman AFM (2003) Assumption-fre analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66CrossRefGoogle Scholar
  37. Repčák M, Pal’ove-Balang P, Dučaiová Z, Sajko M, Bendek F (2014) High nitrogen supply affects the metabolism of Matricaria chamomilla leaves. Plant Growth Regul 73:147–153CrossRefGoogle Scholar
  38. Rogato A, D’Apuzzo E, Barbulova A, Omrane S, Parlati A, Carfagna S, Costa A, Lo Schiavo F, Esposito S, Chiurazzi M (2010) Characterization of a developmental root response caused by external ammonium supply in Lotus japonicus. Plant Physiol 154:784–795CrossRefGoogle Scholar
  39. Salvemini F, Marini AM, Riccio A, Patriarca EJ, Chiurazzi M (2001) Functional characterization of an ammonium transporter gene from Lotus japonicus. Gene 270:237–243CrossRefGoogle Scholar
  40. Schwacke R, Schneider A, van der Graaff E, Fischer K, Catoni E, Desimone M, Frommer WB, Flugge UI, Kunze R (2003) ARAMEMNON, a novel database for Arabidopsis integral membrane proteins. Plant Physiol 131:16–26CrossRefGoogle Scholar
  41. Singh RP, Sainger M, Singh DP, Jaiwal PK (2008) Nitrate and ammonium transporters in plants. Plant membrane and vacuolar transporters. CAB International, Wallingford, pp 345–371Google Scholar
  42. Song ZZ, Su YH (2013) Distinctive potassium-accumulation capability of alligatorweed (Alternanthera philoxeroides) links to high-affinity potassium transport facilitated by K+-uptake systems. Weed Sci 61:77–84CrossRefGoogle Scholar
  43. Song T, Gao Q, Xu Z, Song R (2010) The cloning and characterization of two ammonium transporters in the salt-resistant green alga, Dunaliella viridis. Mol Biol Rep 38:4797–4804CrossRefGoogle Scholar
  44. Song ZZ, Yang SY, Zhu H, Jin M, Su YH (2014) Heterologous expression of an alligatorweed high-affinity potassium transporter gene enhances salinity tolerance in Arabidopsis. Am J Bot 101:840–850CrossRefGoogle Scholar
  45. Sonoda Y, Ikeda A, Saiki S, von Wirén N, Yamaya T, Yamaguchi J (2003a) Distinct expression and function of three ammonium transporter genes (OsAMT1;1–1;3) in rice. Plant Cell Physiol 44:726–734CrossRefGoogle Scholar
  46. Sonoda Y, Ikeda A, Saiki S, Yamaya T, Yamaguchi J (2003b) Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice. Plant Cell Physiol 44:1396–1402CrossRefGoogle Scholar
  47. Suenaga A, Moriya K, Sonoda Y, Ikeda A, von Wirén N, Hayakawa T, Yamaguchi J, Yamaya T (2003) Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants. Plant Cell Physiol 44:206–211CrossRefGoogle Scholar
  48. Syrett PJ, Morris I (1963) The inhibition of nitrate assimilation by ammonium in Chlorella. Biochem Biophys Acta 67:566–575CrossRefGoogle Scholar
  49. von Wirén N, Gazzarini S, Gojon A, Formmer WB (2000) The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol 3:254–261CrossRefGoogle Scholar
  50. Wang MY, Siddiqi MY, Ruth TJ, Glass ADM (1993) Ammonium uptake by rice roots (II. Kinetics of NH4 +-13N- InXux across the plasmalemma). Plant Physiol 103:1259–1267CrossRefGoogle Scholar
  51. Willian JJ (1986) The role of water plant in water treatment. Agric Eng 57:9–10Google Scholar
  52. Yang SY, Hao DL, Cong Y, Jin M, Su YH (2015) The rice OsAMT1;1 is a proton-independent feedback regulated ammonium transporter. Plant Cell Rep 34:321–330CrossRefGoogle Scholar
  53. Yuan L, Loqué D, Ye FW, Frommer B, von Wirén N (2007) Nitrogen-dependent posttranscriptional regulation of the ammonium transporter AtAMT1;1. Plant Physiol 143:732–744CrossRefGoogle Scholar
  54. Yuan L, Graff L, Loque D, Kojima S, Tsuchiya YN, Takahashi H, von Wirén N (2009) AtAMT1.4, a pollen-specific high-affinity ammonium transporter of the plasma membrane in Arabidopsis. Plant Cell Physiol 50:13–25CrossRefGoogle Scholar
  55. Yuan L, Gu R, Xuan Y, Smith-Valle E, Loqué D, Frommer WB, von Wirén N (2013) Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo. Plant Cell 25:974–984CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.College of AgricultureLudong UniversityYantaiChina
  2. 2.Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong (Ludong University)YantaiChina
  3. 3.State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil ScienceChinese Academy of SciencesNanjingChina
  4. 4.College of AgricultureInner Mongolia Agricultural UniversityHuhhotChina

Personalised recommendations