Characterization and expression analysis of BcAMT1;4, an ammonium transporter gene in flowering Chinese cabbage

  • Lihua Zhong
  • Xinmin Huang
  • Yunna Zhu
  • Erfeng Kou
  • Houcheng Liu
  • Guangwen Sun
  • Riyuan Chen
  • Shiwei SongEmail author
Research Report


Ammonium (NH4+) is generated during many endogenous metabolic processes in the leaves of plants, and there is increasing evidence that ammonium transporters (AMTs) play important roles in NH4+ transmembrane transport and distribution. However, the expression of different AMT genes is tissue-type specific and their functions differ. Information about AMT genes and their expression under different environmental conditions in flowering Chinese cabbage (Brassica campestris L.) is currently limited. Here, we isolated and characterized an AMT gene, BcAMT1;4, in flowering Chinese cabbage. BcAMT1;4 was localized to the plasma membrane and complemented NH4+ transport in NH4+ uptake-deficient yeast. The highest expression levels of BcAMT1;4 were detected in the flowers and leaves of flowering Chinese cabbage. The expression of BcAMT1;4 was induced by nitrogen deficiency and significantly inhibited by the reapplication of NH4+ (NH4Cl or NH4NO3). In contrast, when plants pre-cultured in nitrate were transferred to an NH4+ nutrient solution, BcAMT1;4 expression was significantly enhanced. BcAMT1;4 exhibited a diurnal expression pattern, with higher expression levels during the light period than during the dark period, and a peak expression at the later stage of the light period. Knowledge of AMT genes in flowering Chinese cabbage will lay a foundation for enhancing our understanding of the functional roles of different AMT members in the regulation of its growth by NH4+, as BcAMT1;4 seems to play an important role in leaf NH4+ transport.


Ammonium transporters NH4+ Leaf Circadian rhythm Flowering Chinese cabbage 



This work was supported by the National Natural Science Foundation of China [Grant Number 31401855] and the China Agriculture Research System [Grant Number CARS-25-C-04]. We thank Dr. Bruno André from the Université Libre de Bruxelles for kindly providing the ammonium uptake-deficient yeast strain 31019b.

Author contribution

SS conceived and designed the experiments; LZ and XH performed the experiments and wrote the manuscript; YZ and EK completed the yeast functional complementation test. HL helped analyse the data; GS and RC helped to revise the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no competing interests.


  1. Adetunji AT, Lewu FB, Mundembe R (2015) Vigna subterranea, ammonium transporter gene (VsAMT1): some bioinformatics insights. Biotechnol Rep 8:88–93CrossRefGoogle Scholar
  2. Ahmed AK, Johnson KA (2000) The effect of the ammonium: nitrate nitrogen ratio, total nitrogen, salinity (NaCl) and calcium on the oxalate levels of Tetragonia tetragonioides Pallas. Kunz J Hortic Sci Biotechnol 75:533–538CrossRefGoogle Scholar
  3. Alvarez JM, Vidal EA, Gutiérrez RA (2012) Integration of local and systemic signaling pathways for plant N responses. Curr Opin Plant Biol 15:185–191CrossRefGoogle Scholar
  4. Bauwe H (2010) Recent developments in photorespiration research. Biochem Soc Trans 38:677–682CrossRefGoogle Scholar
  5. Bijlsma BJ, Lambers H, Kooijman SALM (2000) A dynamic whole-plant model of integrated metabolism of nitrogen and carbon. 1. Comparative ecological implications of ammonium-nitrate interactions. Plant Soil 220:49–69CrossRefGoogle Scholar
  6. Bittsánszky A, Pilinszky K, Gyulai G, Komives T (2015) Overcoming ammonium toxicity. Plant Sci 231:184–190CrossRefGoogle Scholar
  7. Bloom AJ, Sukrapanna SS, Warner RL (1992) Root respiration associated with ammonium and nitrate absorption and assimilation by barley. Plant Physiol 99:1294–1301CrossRefGoogle Scholar
  8. Bonasia A, Conversa G, Gonnella M, Serio F, Santamaria P (2008) Effects of ammonium and nitrate nutrition on yield and quality in endive. J Hortic Sci Biotechnol 88:64–70CrossRefGoogle Scholar
  9. Britto DT, Kronzucker HJ (2002) NH4 + toxicity in higher plants: a critical review. J Plant Physiol 159:567–584CrossRefGoogle Scholar
  10. Bybordi A (2012) Effect of different ratios of nitrate and ammonium on photosynthesis, and fatty acid composition of canola under saline conditions. Int J Agric Crop Sci 4:622–626Google Scholar
  11. 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
  12. de Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, Bairoch A, Hulo N (2006) ScanProsite: detection of PROSITE signature matches and ProRule-associate functional and structural residues in proteins. Nucleic Acids Res 34:w362–w365CrossRefGoogle Scholar
  13. 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
  14. Gojon A, Nacry P, Davidian JC (2009) Root uptake regulation: a central process for NPS homeostasis in plants. Curr Opin Plant Biol 12:328–338CrossRefGoogle Scholar
  15. Gu R, Duan F, An X, Zhang F, Yuan L (2013) Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.). Plant Cell Physiol 54:1515–1524CrossRefGoogle Scholar
  16. Guan M, Møller IS, Schjoerring JK (2015) Two cytosolic glutamine synthetase isoforms play specific roles for seed germination and seed yield structure in Arabidopsis. J Exp Bot 66:203–212CrossRefGoogle Scholar
  17. Hu L, Yu J, Liao W, Zhang G, Xie J, Lv J, Xiao M, Yang B, Zhou R, Bu R (2015) Moderate ammonium: nitrate alleviates low light intensity stress in mini Chinese cabbage seedling by regulating root architecture and photosynthesis. SciHortic 186:143–153Google Scholar
  18. Huang X, Lei Y, Guan H, Hao Y, Liu H, Sun G, Chen R, Song S (2017) Transcriptomic analysis of the regulation of stalk development in flowering Chinese cabbage (Brassica campestris) by RNA sequencing. Sci Rep 7:15517CrossRefGoogle Scholar
  19. Husted S, Schjoerring JK (1995) A computer-controlled system for studying ammonia exchange, photosynthesis and transpiration of plants canopies growing under controlled environmental conditions. Plant Cell Environ 18:1070–1077CrossRefGoogle Scholar
  20. Husted S, Mattsson M, Möllers C, Wallbraun M, Schjoerring JK (2002) Photorespiratory NH4 + production in leaves of wild-type and glutamine synthetase 2 antisense oilseed rape. Plant Physiol 130:989–998CrossRefGoogle Scholar
  21. Ibrahim A, Jin XL, Zhang YB, Cruz J, Vichyavichien P, Esiobu N, Zhang XH (2017) Tobacco plants expressing the maize nitrate transporter ZmNrt2. 1 exhibit altered responses of growth and gene expression to nitrate and calcium. Bot Stud 58:51CrossRefGoogle Scholar
  22. Jian S, Liao Q, Song H, Liu Q, Lepo JE, Guan C, Zhang J, Zhang Z (2017) NRT1.1-dependent NH4 + toxicity in Arabidopsis is associated with disturbed balance between NH4 + uptake and assimilation.
  23. Kumagai E, Araki T, Hamaoka N, Ueno O (2011) Ammonia emission from rice leaves in relation to photorespiration and genotypic differences in glutamine synthetase activity. Ann Bot 108:1381–1386CrossRefGoogle Scholar
  24. Li H, Cong Y, Chang YH, Lin J (2015) Two AMT2-type ammonium transporters from Pyrus betulaefolia, demonstrate distinct expression characteristics. Plant Mol Biol Rep 34:1–13Google Scholar
  25. Li H, Han JL, Chang YH, Jing L, Yang QS (2016) Gene characterization and transcription analysis of two new ammonium transporters in pear rootstock (Pyrus betulaefolia). J Plant Res 129:737–748CrossRefGoogle Scholar
  26. 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
  27. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  28. Loqué D, von Wirén N (2004) Regulatory levels for the transport of ammonium in plant roots. J Exp Bot 55:1293–1305CrossRefGoogle Scholar
  29. Loqué D, Yuan L, Kojima S, Gojon A, Wirth J, Gazzarrini S, Von Wirén N (2006) Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots. Plant J 48:522–534CrossRefGoogle Scholar
  30. Ludewig U (2006) Ion transport versus gas conduction: function of AMT/Rh-type proteins. Transfus Clin Biol 13:111–116CrossRefGoogle Scholar
  31. Ludewig U, Neuhäuser B, Dynowsky M (2007) Molecular mechanisms of ammonium transport and accumulation in plants. FEBS Lett 581:2301–2308CrossRefGoogle Scholar
  32. Lupini A, Princi MP, Araniti F, Miller AJ, Sunseri F, Abenavoli MR (2017) Physiological and molecular responses in tomato under different forms of n nutrition. J Plant Physiol 216:17CrossRefGoogle Scholar
  33. Marini AM, Urrestarazu A, Beauwens R, Andre B (1997) The Rh (rhesus) blood group polypeptides are related to NH4 + transporters. Trends Biochem Sci 22:460–461CrossRefGoogle Scholar
  34. Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K, Lelandais M, Grandjean O, Kronenberger J, Suzuki A (2006) Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco. Plant Physiol 140:444–456CrossRefGoogle Scholar
  35. Mattsson M, Husted S, Schjoerring JK (1998) Influence of nitrogen nutrition and metabolism on ammonia volatilization in plants. Nutr Cycl Agroecosyst 51:35–40CrossRefGoogle Scholar
  36. McDonald TR, Dietrich FS, Lutzoni F (2012) Multiple horizontal gene transfers of ammonium transporters/ammonia permeases from prokaryotes to eukaryotes: toward a new functional and evolutionary classification. Mol Biol Evol 29:51–60CrossRefGoogle Scholar
  37. Miller AJ, Smith SJ (2008) Cytosolic nitrate ion homeostasis: could it have a role in sensing nitrogen status? Ann Bot 101:485–489CrossRefGoogle Scholar
  38. Nielsen KH, Schjoerring JK (1998) Regulation of apoplastic NH4 + concentration in leaves of oilseed rape. Plant Physiol 118:1361–1368CrossRefGoogle Scholar
  39. Ninnemann O, Jauniaux JC, Frommer WB (1994) Identification of a high affinity NH4 + transporter from plants. EMBO J 13:3464–3471CrossRefGoogle Scholar
  40. Pearson JN, Finnemann J, Schjoerring JK (2002) Regulation of the high-affinity ammonium transporter (BnAMT1; 2) in the leaves of Brassica napus by nitrogen status. Plant Mol Biol 49:483–490CrossRefGoogle Scholar
  41. Rolny N, Bayardo M, Guiamet JJ, Costa L (2016) Nitrogen fertilization increases ammonium accumulation during senescence of barley leaves. Acta Physiol Plant 38:1–11CrossRefGoogle Scholar
  42. Ruamrungsri S, Ruamrungsri S, Ikarashi T, Ohyama T (2000) Ammonium and nitrate assimilation in narcissus roots. J Hortic Sci Biotechnol 75:223–227CrossRefGoogle Scholar
  43. Schjoerring JK, Husted S, Mäck G, Nielsen HH, Finnemann J, Mattsson M (2000) Physiological regulation of plant-atmosphere ammonia exchange. Plant Soil 221:95–102CrossRefGoogle Scholar
  44. Shah K, Gadella TW Jr, van Erp H, Hecht V, de Vries SC (2001) Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein. J Mol Biol 309:641–655CrossRefGoogle Scholar
  45. Sohlenkamp C, Shelden M, Howitt S, Udvardi M (2000) Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. FEBS Lett 476:273–278CrossRefGoogle Scholar
  46. Song SW, Yi LY, Liu HC, Sun GW, Chen RY (2012) Effect of ammonium and nitrate ratios on growth and yield of flowering chinese cabbage. Appl Mech Mater 142:188–192CrossRefGoogle Scholar
  47. Song S, He Z, Huang X, Zhong L, Liu H, Sun G, Chen R (2017a) Cloning and characterization of the ammonium transporter genes BaAMT1;1, and BaAMT1;3, from Chinese kale. Hortic Environ Biotechnol 58:178–186CrossRefGoogle Scholar
  48. Song S, Yi L, Zhu Y, Liu H, Un GS, Chen R (2017b) Effects of ammonium and nitrate ratios on plant growth, nitrate concentration and nutrient uptake in flowering Chinese cabbage. Bangladesh J Bot 46:1259–1267Google Scholar
  49. Sonoda Y, Ikeda A, Saiki S, von Wirén N, Yamaya T, Yamaguchi J (2003) Distinct expression and function of three ammonium transporter genes (OsAMT1; 1-1; 3) in rice. Plant Cell Physiol 44:726–734CrossRefGoogle Scholar
  50. Wang W, Li R, Zhu Q, Tang X, Zhao Q (2016) Transcriptomic and physiological analysis of common duckweed Lemna minor responses to NH4 + toxicity. BMC Plant Biol 16:92CrossRefGoogle Scholar
  51. Yuan L, Loqué D, Kojima S, Rauch S, Ishiyama K, Inoue E, von Wirén N (2007) The organization of high-affinity ammonium uptake in Arabidopsis roots depends on the spatial arrangement and biochemical properties of AMT1-type transporters. Plant Cell 19:2636–2652CrossRefGoogle Scholar
  52. Yuan L, Graff L, Loqué 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
  53. Zhang ZH, Huang HT, Song HX, Liu Q, Rong XM, Peng JW, Guan CY (2012) Research advances on nitrate nitrogen reutilization by proton pump of tonoplast and its relation to nitrogen use efficiency. Aust J Crop Sci 6:1377Google Scholar
  54. Zhang F, Liu Y, Wang L, Bai P, Ruan L, Zhang C, Wei K, Cheng H (2018) Molecular cloning and expression analysis of ammonium transporters in different tea (Camellia sinensis (l.) o. kuntze) cultivars under different nitrogen treatments. Gene 658:136–145CrossRefGoogle Scholar
  55. Zhong L, Zhang Y, Liu H, Sun G, Chen R, Song S (2016) Agrobacterium-mediated transient expression via root absorption in flowering Chinese cabbage. SpringerPlus 5:1825CrossRefGoogle Scholar
  56. Zhu Y, Hao Y, Liu H, Sun G, Chen R, Song S (2018) Identification and characterization of two ammonium transporter genes in flowering Chinese cabbage (Brassica campestris). Plant Biotechnol 35:59–70CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2019

Authors and Affiliations

  • Lihua Zhong
    • 1
    • 2
  • Xinmin Huang
    • 1
  • Yunna Zhu
    • 1
  • Erfeng Kou
    • 1
  • Houcheng Liu
    • 1
  • Guangwen Sun
    • 1
  • Riyuan Chen
    • 1
  • Shiwei Song
    • 1
    Email author
  1. 1.College of HorticultureSouth China Agricultural UniversityGuangzhouChina
  2. 2.College of Agriculture and Food EngineeringBaise UniversityBaiseChina

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