Skip to main content
Log in

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S521) were placed in darkness in a buffer containing 50 mM KNO3, nitrite was excreted from the tissue at rates of 0.08–0.2 μmol (g FW)−1 h−1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S521, although 20–40 μmol (g FW)−1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S521 also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S521 was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1a–d
Fig. 2
Fig. 3a–d
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

NR :

Nitrate reductase

NO :

Nitric oxide

Ser :

Serine

WT :

Wild type

References

  • Allègre A (2003) Approche physiologique et biomoléculaire du rôle de la nitrate réductase dans la résistance à l’asphyxie racinaire de la tomate. Thesis, Institut National Polytechnique de Toulouse, France

  • Athwal GS, Huber SC (2002) Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase. Plant J 29:119–129

    Article  CAS  PubMed  Google Scholar 

  • Bachmann M, Shiraishi N, Campbell WH, Yoo B-C, Harmon AC, Huber SC (1996) Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell 8:505–517

    Article  CAS  PubMed  Google Scholar 

  • Botrel A, Kaiser WM (1997) Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status. Planta 201:496–501

    Article  CAS  PubMed  Google Scholar 

  • Botrel A, Magné C, Kaiser WM (1996) Nitrate reduction, nitrite reduction and ammonia assimilation in barley roots in response to anoxia. Plant Physiol Biochem 34:645–652

    CAS  Google Scholar 

  • Dordas C, Hasinoff BB, Igamberdiev AU, Manac’h N, Rivoal J, Hill RD (2003) Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa root cultures under hypoxic stress. Plant J 35:763–770

    Article  CAS  PubMed  Google Scholar 

  • Douglas P, Morrice N, MacKintosh C (1995) Identification of a regulatory phosphorylation site in the hinge 1 region of nitrate reductase from spinach (Spinacea oleracea) leaves. FEBS Lett 377:113–117

    Article  CAS  PubMed  Google Scholar 

  • Dry I, Wallace W, Nicholas DJD (1981) Role of ATP in nitrite reduction in roots of wheat and pea. Planta 152:234–238

    CAS  Google Scholar 

  • Ferrari TE, Yoder OC, Filner P (1973) Anaerobic nitrite production by plant cells and tissues: evidence for two nitrate pools. Plant Physiol 51:423–431

    CAS  Google Scholar 

  • Huber SC, MacKintosh C, Kaiser WM (2002) Metabolic enzymes as targets for 14-3-3 proteins. Plant Mol Biol 50:1053–1063

    Article  CAS  PubMed  Google Scholar 

  • Kaiser WM, Kandlbinder A, Stoimenova M, Glaab J (2000) Discrepancy between nitrate reduction rates in intact leaves and nitrate reductase in leaf extracts: What limits nitrate reduction in situ? Planta 210:801–807

    CAS  PubMed  Google Scholar 

  • Kaiser WM, Planchet E, Stoimenova M, Sonoda M (2004) Modulation of nitrate reductase activity and (eco)physiological implications. In: Stulen I, Amancio Z (eds) N metabolism and plant adaption to the environment, chapter 7. Kluwer, Dordrecht

  • Lee RB (1979) The release of nitrite from barley roots in response to metabolic inhibitors, uncoupling agents and anoxia. J Exp Bot 30:119–133

    CAS  Google Scholar 

  • Leprince A-S, Grandbastien M-A, Meyer C (2001) Retrotransposons of the Tnt1B family are mobile in Nicotiana plumbaginifolia and can induce alternative splicing of the host gene upon insertion. Plant Mol Biol 47:533–541

    Article  CAS  PubMed  Google Scholar 

  • Lillo C (2004) Light regulation of nitrate uptake, assimilation and metabolism. In: Stulen I, Amancio Z (eds) N metabolism and plant adaption to the environment, chapter 6. Kluwer, Dordrecht

  • Lillo C, Henriksen A (1984) Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley. Physiol Plant 62:89–94

    CAS  Google Scholar 

  • Lillo C, Kazaic S, Ruoff P, Meyer C (1997) Characterization of nitrate reductase from light- and dark-exposed leaves. Plant Physiol 114:1377–1383

    CAS  PubMed  Google Scholar 

  • Lillo C, Lea US, Leydecker M-T, Meyer C (2003) Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation. Plant J 35:566–573

    Article  CAS  Google Scholar 

  • Mann AF, Hucklesby DP, Hewitt EJ (1979) Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves. Planta 146:83–89

    CAS  Google Scholar 

  • Martinoia E, Heck U, Wiemken A (1981) Vacuoles as storage compartment for nitrate in barley leaves. Nature 289:292–294

    CAS  Google Scholar 

  • Meyer C, Stitt M (2001) Nitrate reduction and signalling. In: Lea PJ, Morot-Gaudry JF (eds) Plant nitrogen. Springer, Berlin Heidelberg New York, pp 37–59

  • Morot-Gaudry-Talamain Y, Rockel P, Moureaux T, Quilleré I, Leydecker M-T, Kaiser WM, Morot-Gaudry JF (2002) Nitrite accumulation and NO emission in relation to cellular signaling in NiR antisense tobacco. Planta 215:708–715

    Article  PubMed  Google Scholar 

  • Provan F, Aksland L-M, Meyer C, Lillo C (2000) Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties. Plant Physiol 123:757–764

    CAS  PubMed  Google Scholar 

  • Riens B, Heldt HW (1992) Decrease of nitrate reductase activity in spinach leaves during a light–dark transition. Plant Physiol 98:573–577

    CAS  Google Scholar 

  • Rockel P, Strube F, Rockel A, Wildt J, Kaiser WM (2002) Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. J Exp Bot 53:103–110

    Google Scholar 

  • Ruoff P, Lillo C (1990) Molecular oxygen as electron acceptor in the NADH-nitrate reductase system. Biochem Biophys Res Commun 172:1000–1005

    CAS  PubMed  Google Scholar 

  • Sanchez J, Heldt HW (1990) On the regulation of spinach nitrate reductase. Plant Physiol 92:684–689

    CAS  Google Scholar 

  • Stoimenova M, Hänsch R, Mendel R, Gimmler H, Kaiser WM (2003a) The role of nitrate reduction in the anoxic metabolism of roots. I. Characterization of root morphology and normoxic metabolism of wild type tobacco and a transformant lacking root nitrate reductase. Plant Soil 253:145–153

    Article  CAS  Google Scholar 

  • Stoimenova M, Liboureligl, Ratcliffe RG, KaiserWM (2003b) The role of nitrate reduction in the anoxic metabolism of roots. II. Anoxic metabolism of tobacco roots with or without nitrate reductase activity. Plant Soil 253:155–167

    Article  CAS  Google Scholar 

  • Su W, Huber SC, Crawford NM (1996) Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase. Plant Cell 8:519–527

    Google Scholar 

  • van der Leij M, Smith SJ, Miller AJ (1998) Remobilisation of vacuolar stored nitrate in barley root cells. Planta 205:64–72

    Article  Google Scholar 

  • Vincentz M, Caboche M (1991) Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants EMBO J 10:1027–1035

    CAS  Google Scholar 

  • Yamasaki H, Sakihama Y (2000) Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species. FEBS Lett 468:89–92

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the Marie Curie Host Fellowship QLK-3-CT-2001-60058 at the UNAP (U.S.L.), the Socrates/Erasmus program (F.tH.) and the Norwegian Research Council (F.P.)

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Cathrine Lillo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lea, U.S., ten Hoopen, F., Provan, F. et al. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue. Planta 219, 59–65 (2004). https://doi.org/10.1007/s00425-004-1209-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-004-1209-6

Keywords

Navigation