Plant and Soil

, Volume 357, Issue 1–2, pp 205–214 | Cite as

Stimulation of phosphorus uptake by ammonium nutrition involves plasma membrane H+ ATPase in rice roots

  • Houqing Zeng
  • Gan Liu
  • Toshinori Kinoshita
  • Ruiping Zhang
  • Yiyong Zhu
  • Qirong Shen
  • Guohua Xu
Regular Article



Nitrogen, especially NH 4 + , can stimulate the uptake of phosphorus in plants, but the underlying mechanisms have not been clearly elucidated. Because phosphate is taken up via an anion/H+ co-transport process, we propose that the stimulated uptake of phosphorus by NH 4 + versus NO 3 - nutrition may be related to the activity of plasma membrane H+ ATPase. In the present study, we investigated the effect of NH 4 + and NO 3 - on phosphorus uptake and plasma membrane H+ ATPase activity in rice.


Rice plants were cultivated in a hydroponic solution with NH 4 + or NO 3 - . After 15 days of cultivation, phosphorus content was determined. Root plasma membrane was isolated using a two-phase partitioning system and hydrolytic H+-ATPase activity was determined by measuring the Pi concentration after a 30-min hydrolysis reaction. Relative expression of plasma membrane H+ ATPase genes was analyzed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). H+ ATPase enzyme concentration in the plasma membrane was detected by western blot. For 33P uptake experiments, rice roots were incubated in the nutrient solution with addition of H 3 33 PO4.


P content in both the roots and shoots of rice plants supplemented with NH 4 + was significantly higher than P content in plants grown with NO 3 - . Plasma membrane H+ ATPase activity in NH 4 + -fed rice roots was significantly higher than that in NO 3 - -fed rice roots. Real-time qRT-PCR and western blot results indicated that the higher activity of plasma membrane H+ ATPase in NH 4 + -fed rice roots could be attributed to increased expression of the OSA1, OSA3, OSA7, OSA8 and OSA9 genes and an increase in H+ ATPase enzyme concentration in the plasma membrane. Results from 33P uptake experiments showed that rice roots incubated with NH 4 + absorbed more 33P during the four-hour incubation than did rice roots incubated with NO 3 - . Vanadate inhibited 33P uptake in rice roots supplied with NH 4 + , while fusicoccin stimulated 33P uptake under NO 3 - nutrition.


Taken together, these results suggest an involvement of plasma membrane H+ ATPase in the stimulated uptake of phosphorus by rice roots supplemented with NH 4 + .


Ammonium Nitrate Phosphorus uptake Plasma membrane H+ ATPase Rice (Oryza sativa L.) 



This work was supported by Natural Science Foundation of China (NSFC 30971864).


  1. Arango M, Gevaudant F, Oufattole M, Boutry M (2003) The plasma membrane proton pump atpase: the significance of gene subfamilies. Planta 216:355–365PubMedGoogle Scholar
  2. Baginski ES, Foa PP, Zak B (1967) Determination of phosphate: study of labile organic phosphate interference. Clinica Chimica Acta 15:155–158CrossRefGoogle Scholar
  3. Bieleski RL (1973) Phosphate pools, phosphate transport, and phosphate availability. Annu Rev Plant Physiol 24:225–252CrossRefGoogle Scholar
  4. Blair GJ, Mamaril CP, Miller MH (1971) Influence of nitrogen source on phosphorous uptake by corn from soils differing in pH. Agron J 63:235–238CrossRefGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Chang CR, Hu YB, Sun SB, Zhu YY, Ma GJ, Xu GH (2009) Proton pump OsA8 is linked to phosphorus uptake and translocation in rice. J Exp Bot 60:557–565PubMedCrossRefGoogle Scholar
  7. Chiou TJ, Lin SI (2011) Signaling network in sensing phosphate availability in plants. Annu Rev Plant Biol 62:185–206PubMedCrossRefGoogle Scholar
  8. Daram P, Brunner S, Persson BL, Amrhein N, Bucher M (1998) Functional analysis and cell-specific expression of phosphate transporter from tomato. Planta 206:225–233PubMedCrossRefGoogle Scholar
  9. Duan YH, Zhang YL, Ye LT, Fan XR, Xu GH, Shen QR (2007) Responses of rice cultivars with different nitrogen use efficiency to partial nitrate nutrition. Ann Bot 99:1153–1160PubMedCrossRefGoogle Scholar
  10. Duby G, Boutry M (2009) The plant plasma membrane proton pump ATPase: a highly regulated P-type ATPase with multiple physiological roles. Pflugers Archiev – European. J Physiol 457:645–655Google Scholar
  11. Falkengren-Grerup U, Mansson KF, Olsson MO (2000) Uptake capacity of amino acids by ten grasses and forbs in relation to soil acidity and nitrogen availability. Environ Exp Bot 44:207–219PubMedCrossRefGoogle Scholar
  12. Gahoonia TS, Claassen N, Jungk A (1992) Mobilization of phosphate in different soils by ryegrass supplied with ammonium or nitrate. Plant Soil 140:241–248CrossRefGoogle Scholar
  13. Grunes DL (1959) Effect of nitrogen on the availability of soil and fertilizer phosphorous to plants. Adv Agron 11:369–396CrossRefGoogle Scholar
  14. Hayashi Y, Nakamura S, Takemiya A, Takahashi Y, Shimazaki K, Kinoshita T (2010) Biochemical characterization of in vitro phosphorylation and dephosphorylation of the plasma membrane H+-ATPase. Plant Cell Physiol 51:1186–1196PubMedCrossRefGoogle Scholar
  15. Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195CrossRefGoogle Scholar
  16. Hoffmann C, Ladewig E, Claassen N, Jungk A (1994) Phosphorus uptake of maize as by affected ammonium or nitrate nitrogen-measurements and model calculations. Z Pflanzenernahr Bodenk 157:225–232CrossRefGoogle Scholar
  17. Jia H, Ren H, Gu M, Zhao J, Sun S, Zhang X, Chen J, Wu P, Xu G (2011) The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice. Plant Physiol 156:1164–1175PubMedCrossRefGoogle Scholar
  18. Jing J, Rui Y, Zhang F, Rengel Z, Shen J (2010) Localized application of phosphorus and ammonium improves growth of maize seedlings by stimulating root proliferation and rhizosphere acidification. Field Crops Res 119:355–364CrossRefGoogle Scholar
  19. Kant S, Peng M, Rothstein SJ (2011) Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis. PLoS Genetics 7:e1002021PubMedCrossRefGoogle Scholar
  20. Kinoshita T, Hayashi Y (2011) New insights into the regulation of stomatal opening by blue light and plasma membrane H+-ATPase. Int Rev Cell Mol Biol 289:89–115PubMedCrossRefGoogle Scholar
  21. Kronzucker HJ, Siddiqi MY, Glass ADM (1997) Conifer root discrimination against soil nitrate and the ecology of forest succession. Nature 385:59–61CrossRefGoogle Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  23. Lamaze T, Sentenac H, Grignon C (1984) Effects of nitrate on phosphate accumulation and transport by corn roots. Physiol Veg 22:155–161Google Scholar
  24. Larsson C (1985) Plasma membrane. In: Linskens HF, Jackson JF (eds) Modern methods of plant analysis. Springer-Verlag, Berlin, pp 85–104Google Scholar
  25. Li B, Wang S, Feng H, Xu G (2008) Effects of nitrogen forms on root morphology and phosphate uptake in rice. Chinese J Rice Sci 22(5):665–668 (in Chinese)Google Scholar
  26. Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic, London, pp 231–254Google Scholar
  27. Marschner H, Römheld V (1983) In vivo measurement of root induced pH changes at the soil-root interface: effect of plant species and nitrogen source. Zeitschrift für Panzenphysiologie und Bodenkunde 111:241–251Google Scholar
  28. Mengel K, Schubert S (1985) Active extrusion of protons into deionized water by roots of intact maize plants. Plant Physiol 79:344–348PubMedCrossRefGoogle Scholar
  29. Miller MH (1974) Effect of nitrogen on phosphorus absorption by plants. In: Carson W (ed) The plant root and its environment. Univ. Press of Virginia, Charlottesville, pp 643–668Google Scholar
  30. Mimura T (1999) Regulation of phosphate transport and homeostasis in plant cells. Int Rev Cytol 191:149–200CrossRefGoogle Scholar
  31. Mistrik I, Ullrich C (1996) Mechanism of anion uptake in plant roots: quantitative evaluation of H+/NO3- and H+/H2PO4- stoichiometries. Plant Physiol Biochem 34:629–636Google Scholar
  32. Mitsukawa N, Okumura S, Shirano Y, Sato S, Kato T, Harashima S, Shibata D (1997) Overexpression of an Arabidopsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances cell growth under phosphate-limited conditions. Proc Natl Acad Sci U S A 94:7098–7102PubMedCrossRefGoogle Scholar
  33. Moorby H, Nye P, White R (1985) The influence of nitrate nutrition on H+ efflux by young rape plants (Brassica napus cv. emerald). Plant Soil 84:403–415CrossRefGoogle Scholar
  34. Ortas I, Harris EJ, Rowell DL (1996) Enhanced uptake of phosphorus by mycorrhizal sorghum plants as influenced by forms of nitrogen. Plant Soil 184:255–264CrossRefGoogle Scholar
  35. Palmgren MG (1998) Proton gradient and plant growth: role of the plasma membrane H+-ATPase. Adv Bot Res 28:1–70CrossRefGoogle Scholar
  36. Palmgren M, Harper J (1999) Pumping with plant P-type ATPases. J Exp Bot 50:883–893Google Scholar
  37. Preuss CP, Huang CY, Tyerman SD (2011) Proton-coupled high-affinity phosphate transport revealed from heterologous characterization in Xenopus of barley-root plasma membrane transporter, HvPHT1;1. Plant Cell Environ 34:681–689PubMedCrossRefGoogle Scholar
  38. Rae AL, Cybinski DH, Jarmey JM, Smith FW (2003) Characterization of two phosphate transporters frombarley; evidence for diverse function and kinetic properties among members of the Pht1 family. Plant Mol Biol 53:27–36PubMedCrossRefGoogle Scholar
  39. Raghothama KG, Karthikeyan AS (2005) Phosphate acquisition. Plant Soil 274:37–49CrossRefGoogle Scholar
  40. Richardson AE (2009) Regulating the phosphorus nutrition of plants: molecular biology meeting agronomic needs. Plant Soil 322:17–24CrossRefGoogle Scholar
  41. Riley D, Barber SA (1971) Effect of ammonium and nitrate fertilization on phosphorous uptake as related to root-induced pH changes at the root-soil interface. Soil Sci Soc Am 35:301–306CrossRefGoogle Scholar
  42. Sakano K, Yazaki Y, Mimura T (1992) Cytoplasmic acidification induced by inorganic phosphate uptake in suspension cultured Catharanthus roseus cells. Plant Physiol 99:672–680PubMedCrossRefGoogle Scholar
  43. Sarkar AN, Wyn Jones RGW (1982) Influence of rhizosphere on the nutrient status of dwarf French beans. Plant Soil 64:369–380CrossRefGoogle Scholar
  44. Schachtman DP, Reid RJ, Ayling SM (1998) Phosphorus uptake by plants: from soil to cell. Plant Physiol 116:447–453PubMedCrossRefGoogle Scholar
  45. Schaller G, Fischer WR (1985) pH-Änderungen in der Rhizoshpare von Mais und Erdnuswurzeln. Zeitschrift für Panzenphysiologie und Bodenkunde 148:306–320CrossRefGoogle Scholar
  46. Schubert S, Yan F (1997) Nitrate and ammonium nutrition of plants: effects on acid/base balance and adaptation of root cell plasmalemma H+-ATPase. Zeitschrift für Panzenphysiologie und Bodenkunde 160:275–281CrossRefGoogle Scholar
  47. Serrano R (1989) Structure and function of plasma membrane ATPase. Annu Rev Plant Physiol Plant Mol Biol 40:61–94CrossRefGoogle Scholar
  48. Shen H, Chen J, Wang Z, Yang C, Sasaki T, Yamamoto Y, Matsumoto H, Yan X (2006) Root plasma membrane H+-ATPase is involved in the adaptation of soybean to phosphorus starvation. J Exp Bot 57:1353–1362PubMedCrossRefGoogle Scholar
  49. Smith FW (2001) Sulphur and phosphorus transport systems in plants. Plant Soil 232:109–118CrossRefGoogle Scholar
  50. Smith FW, Jackson WA (1987) Nitrogen enhancement of phosphate transport in roots of Zea mays L. I. Effects of ammonium and nitrate pretreatment. Plant Physiol 84:1314–1318PubMedCrossRefGoogle Scholar
  51. Song W, Makeen K, Wang D, Zhang C, Xu Y, Zhao H, Tu E, Zhang Y, Shen Q, Xu G (2011) Nitrate supply affects root growth differentially in two rice cultivars differing in nitrogen use efficiency. Plant Soil 343:357–368CrossRefGoogle Scholar
  52. Sze H, Li X, Palmgren MG (1999) Energization of plant cell membranes by H+-pumping ATPases: regulation and biosynthesis. Plant Cell 11:677–689PubMedGoogle Scholar
  53. Thien SJ, Mcfee WW (1972) Effect of nitrogen on phosphorous transport systems in Zea mays L. Proc Soil Sci Soc Am 36:617–620CrossRefGoogle Scholar
  54. Ullrich CI, Novacky AJ, Fisher E, Liittge U (1981) Relationship between energy-dependent phosphate-uptake and the electrical membrane-potential in Lemna gibba L. Plant Physiol 67:797–801CrossRefGoogle Scholar
  55. Ullrich CI, Novacky AJ, van Bell AJE (1984) Phosphate uptake in Lemna gibba G1: energetics and kinetics. Planta 161:45–52Google Scholar
  56. Wang MY, Siddiqi MY, Ruth TJ, Glass A (1993) Ammonium uptake by rice roots. II. Kinetics of 13NH4+ influx across the plasmalemma. Plant Physiol 103:1259–1267PubMedCrossRefGoogle Scholar
  57. Wiersum LK (1958) Density of root branching as affected by substrate and separate ions. Acta Bot Neerl 7:174–190Google Scholar
  58. Yamaya T, Oaks A (2004) Metabolic regulation of ammonium uptake and assimilation. In: Amancio S, Stulen I (eds) Nitrogen Acquisition and Assimilation in Higher Plants (Plant Ecophysiology Series) (Kluwer Academic Publisher, Dordrecht, the Netherlands, pp 35–64Google Scholar
  59. Yan F, Zhu Y, Muller C, Zorb C, Schubert S (2002) Adaptation of H+-pumping and plasma membrane H+-ATPase activity in proteoid roots of white lupin under phosphate deficiency. Plant Physiol 129:50–63PubMedCrossRefGoogle Scholar
  60. Zhang R, Liu G, Wu N, Gu M, Zeng H, Zhu Y, Xu G (2011) Adaptation of plasma membrane H+ ATPase and H+ pump to P deficiency in rice roots. Plant Soil 349:3–11CrossRefGoogle Scholar
  61. Zhu Y, Di T, Xu G, Chen X, Zeng H, Yan F, Shen Q (2009) Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition. Plant Cell Environ 32:1428–1440PubMedCrossRefGoogle Scholar
  62. Zhu Y, Lian J, Zeng H, Liu G, Di T, Shen Q, Xu G (2011) Involvement of plasma membrane H+ ATPase in the adaption of rice to ammonium nutrient. Rice Science 18:335–342CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Houqing Zeng
    • 1
  • Gan Liu
    • 1
  • Toshinori Kinoshita
    • 2
  • Ruiping Zhang
    • 1
  • Yiyong Zhu
    • 1
  • Qirong Shen
    • 1
  • Guohua Xu
    • 1
  1. 1.College of Resources and Environmental SciencesNanjing Agricultural UniversityNanjingChina
  2. 2.Division of Biological Science, Graduate School of ScienceNagoya UniversityNagoyaJapan

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