Molecular Genetics and Genomics

, Volume 278, Issue 2, pp 125–133 | Cite as

Virus-induced gene silencing of 14-3-3 genes abrogates dark repression of nitrate reductase activity in Nicotiana benthamiana

  • Tatsuya HiranoEmail author
  • Akiko Ito
  • Thomas Berberich
  • Ryohei Terauchi
  • Hiromasa Saitoh
Original Paper


In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.


14-3-3 proteins Nicotiana benthamiana Nitrate reductase Virus-induced gene silencing 



We thank Dr David C. Baulcombe (Sainsbury Laboratory, John Innes Centre, Norwich, UK) for the kind gift of plasmid pPC2S and pTXS.GFP. We are grateful to Dr Carol MacKintosh and Dr Jean Harthill (University of Dundee, Dundee, UK) for the generous gift of antiserum to spinach 14-3-3 protein. AI, TB, RT and HS thank a support from “Program for Promotion of Basic Research Activities for Innovative Biosciences” (Japan).


  1. 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 29:119–129PubMedCrossRefGoogle Scholar
  2. Athwal GS, Huber JL, Huber SC (1998) Biological significance of divalent metal ion binding to 14-3-3 proteins in relationship to nitrate reductase inactivation. Plant Cell Physiol 39:1065–1072PubMedGoogle Scholar
  3. Bachmann M, Huber JL, Liao P-C, Gage DA, Huber SC (1996) The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein. FEBS Lett 387:127–131PubMedCrossRefGoogle Scholar
  4. Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2:109–113PubMedCrossRefGoogle Scholar
  5. Baulcombe DC, Chapman S, Santa Crus S (1995) Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7:1045–1053PubMedCrossRefGoogle Scholar
  6. Burton RA, Gibeaut DM, Bacic A, Findlay K, Roberts K, Hamilton A, Baulcombe DC, Fincher GB (2000) Virus-induced silencing of a plant cellulose synthase gene. Plant Cell 12:691–705PubMedCrossRefGoogle Scholar
  7. Chen Z, Fu H, Liu D, Chang PL, Narasimhan M, Ferl R, Hasegawa PM, Bressan RA (1994) A NaCl-regulated plant gene encoding a brain protein homolog that activates ADP ribosyltransferase and inhibits protein kinase C. Plant J 6:729–740PubMedCrossRefGoogle Scholar
  8. Chung H-J, Sehnke PC, Ferl RJ (1999) The 14-3-3 proteins: cellular regulators of plant metabolism. Trends Plant Sci 4:367–371PubMedCrossRefGoogle Scholar
  9. DeLille JM, Sehnke PC, Ferl RJ (2001) The Arabidopsis 14-3-3 family of signaling regulators. Plant Physiol 126:35–38PubMedCrossRefGoogle Scholar
  10. Hageman RH, Reed AJ (1980) Nitrate reductase from higher plants. Methods Enzymol 69:270–280CrossRefGoogle Scholar
  11. Huber JL, Huber SC, Campbell WH, Redinbaugh MG (1992) Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation. Arch Biochem Biophys 296:58–65PubMedCrossRefGoogle Scholar
  12. Huber SC, Bachmann M, Huber JL (1996) Post-translational regulation of nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins. Trends Plant Sci 1:432–438CrossRefGoogle Scholar
  13. Isaacson T, Damasceno CMB, Saravanan RS, He Y, Catalá C, Saladié M, Rose JKC (2006) Sample extraction techniques for enhanced proteomic analysis of plant tissue. Nature Protocol 2:769–774Google Scholar
  14. Kaiser WM, Huber SC (1994) Posttranslational regulation of nitrate reductase in higher plants. Plant Physiol 106:817–821PubMedGoogle Scholar
  15. Kaiser WM, Spill D, Brendle-Behnisch E (1992) Adenine nucleotides are apparently involved in the light–dark modulation of spinach-leaf nitrate reductase. Planta 186:236–240CrossRefGoogle Scholar
  16. Kaiser WM, Spill D, Glaab J (1993) Rapid modulation of nitrate reductase in leaves and roots: indirect evidence for the involvement of protein phosphorylation/dephosphorylation. Physiol Plant 89:557–562CrossRefGoogle Scholar
  17. 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 35:566–573PubMedCrossRefGoogle Scholar
  18. Lillo C, Meyer C, Lea US, Provan F, Oltedal S (2004) Mechanism and importance of post-translational regulation of nitrate reductase. J Exp Bot 55:1275–1282PubMedCrossRefGoogle Scholar
  19. Lu G, Sehnke PC, Ferl RJ (1994) Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog. Plant Cell 6:501–510PubMedCrossRefGoogle Scholar
  20. Lukaszewicz M, Matysiak-Kata I, Aksamit A, Szopa J (2002) 14-3-3 protein regulation of the antioxidant capacity of transgenic potato tubers. Plant Sci 163:125–130CrossRefGoogle Scholar
  21. MacKintosh C (1992) Regulation of spinach-leaf nitrate reductase by reversible phosphorylation. Biochim Biophys Acta 1137:121–126PubMedCrossRefGoogle Scholar
  22. MacKintosh C, Douglas P, Lillo C (1995) Identification of a protein that inhibits the phosphorylated form of nitrate reductase from spinach (Spinacia oleracea) leaves. Plant Physiol 107:451–457PubMedGoogle Scholar
  23. Moore BW, Perez VJ (1967) Specific acidic proteins of the nervous system. In: Carlson FD (eds) Physiological and biochemical aspects of nervous integration. Prentice-Hall, Englewood Cliffs, pp 343–359Google Scholar
  24. Moorhead G, Douglas P, Morrice N, Scarabal M, Aitken A, MacKintosh C (1996) Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activates by fusicoccin. Curr Biol 6:1104–1113PubMedCrossRefGoogle Scholar
  25. Moorhead G, Douglas P, Cotelle V, Harthill J, Morrice N, Meek S, Deiting U, Stitt M, Scarabel M, Aitken A, MacKintosh C (1999) Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins. Plant J 18:1–12PubMedCrossRefGoogle Scholar
  26. Muslin AJ, Tanner JW, Allen PM, Shaw AS (1996) Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84:889–897PubMedCrossRefGoogle Scholar
  27. Piotrowski M, Oecking C (1998) Five new 14-3-3 isoforms from Nicotiana tabacum L.: implications for the phylogeny of plant 14-3-3 proteins. Planta 204:127–130PubMedCrossRefGoogle Scholar
  28. Roberts MR (2000) Regulatory 14-3-3 protein–protein interactions in plant cells. Curr Opin Plant Biol 3:400–405PubMedCrossRefGoogle Scholar
  29. Rosenquist M, Alsterfjord M, Larsson C, Sommarin M (2001) Data mining the Arabidopsis genome reveals fifteen 14-3-3 genes. Expression is demonstrated for two out of five novel genes. Plant Physiol 127:142–149PubMedCrossRefGoogle Scholar
  30. Saitoh H, Terauchi R (2002) Virus-induced silencing of FtsH gene in Nicotiana benthamiana causes a striking bleached leaf phenotype. Genes Genet Syst 77:335–340PubMedCrossRefGoogle Scholar
  31. Saitoh H, Kiba A, Nishihara M, Yamamura S, Suzuki K, Terauchi R (2001) Production of antimicrobial defensin in Nicotiana benthamiana with a potato virus X vector. Mol Plant Microbe Interact 14:111–115PubMedCrossRefGoogle Scholar
  32. Sehnke PC, Chung HJ, Wu K, Ferl RJ (2001) Regulation of starch accumulation by granule-associated plant 14-3-3 proteins. Proc Natl Acad Sci USA 98:765–770PubMedCrossRefGoogle Scholar
  33. Sehnke PC, DeLille JM, Ferl RJ (2002) Consummating signal transduction: the role of 14-3-3 proteins in the completion of signal-induced transitions in protein activity. Plant Cell 14(Suppl):S339–S354PubMedGoogle Scholar
  34. Sinnige MP, Roobeek I, Bunney TD, Visser AJWG, Mol JNM, de Boer AH (2005) Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase. Plant J 44:1001–1009PubMedCrossRefGoogle Scholar
  35. 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 8:519–527PubMedCrossRefGoogle Scholar
  36. Thomas CL, Jones L, Baulcombe DC, Maule AJ (2001) Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vector. Plant J 25:417–425PubMedCrossRefGoogle Scholar
  37. Voinnet O (2001) RNA silencing as a plant immune system against viruses. Trends Genet 17:449–459PubMedCrossRefGoogle Scholar
  38. Wilczynski G, Kulma A, Szopa J (1998) The expression of 14-3-3 isoforms in potato is developmentally regulated. J Plant Physiol 153:118–126Google Scholar
  39. Yan J, He C, Wang J, Mao Z, Holaday SA, Allen RD, Zhang H (2004) Overexpression of the Arabidopsis 14-3-3 protein GF14λ in cotton leads to a “stay-green” phenotype and improves stress tolerance under moderate drought conditions. Plant Cell Physiol 45:1007–1014PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Tatsuya Hirano
    • 1
    Email author
  • Akiko Ito
    • 2
  • Thomas Berberich
    • 2
  • Ryohei Terauchi
    • 2
  • Hiromasa Saitoh
    • 2
  1. 1.Faculty of AgricultureMeijo UniversityNagoyaJapan
  2. 2.Iwate Biotechnology Research CenterKitakamiJapan

Personalised recommendations