Plant Molecular Biology

, Volume 81, Issue 1–2, pp 13–25 | Cite as

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

  • Venkatasalam Shanmugabalaji
  • Céline Besagni
  • Lucia Eugeni Piller
  • Veronique Douet
  • Stephanie Ruf
  • Ralph Bock
  • Felix Kessler
Article

Abstract

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35–HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35–HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.

Keywords

Plastid transformation Plastoglobule Molecular farming HIVp24 HCV core protein Nicotiana tabacum 

Supplementary material

11103_2012_9977_MOESM1_ESM.pptx (2.4 mb)
Supplementary material 1 (PPTX 2485 kb)

References

  1. Arnon DI (1949) Copper enzymes in isolated chloroplasts—polyphenoloxidase in beta-vulgaris. Plant Phys 24:1–15CrossRefGoogle Scholar
  2. Austin JR, Frost E, Vidi PA, Kessler F, Staehelin LA (2006) Plastoglobules are lipoprotein subcompartments of the chloroplast that are permanently coupled to thylakoid membranes and contain biosynthetic enzymes. Plant Cell 18:1693–1703PubMedCrossRefGoogle Scholar
  3. Bally J, Paget E, Droux M, Job C, Jo D, Dubald M (2008) Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins. Plant Biotech J 6:46–61Google Scholar
  4. Birch-Machin I, Newell CA, Hibberd JM, Gray JC (2004) Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability. Plant Biotech J 2:261–270CrossRefGoogle Scholar
  5. Bock R (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol 312:425–438PubMedCrossRefGoogle Scholar
  6. Bondada BR, Syvertsen JP (2003) Leaf chlorophyll, net gas exchange and chloroplast ultrastructure in citrus leaves of different nitrogen status. Tree Physiol 23:553–559PubMedCrossRefGoogle Scholar
  7. Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed-based expression systems for plant molecular farming. Plant Biotech J 8:588–606CrossRefGoogle Scholar
  8. Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J 27:171–178PubMedCrossRefGoogle Scholar
  9. De Marchis F, Pompa A, Mannucci R, Morosinotto T, Bellucci M (2011) A plant secretory signal peptide targets plastome-encoded recombinant proteins to the thylakoid membrane. Plant Mol Biol 76:427–441PubMedCrossRefGoogle Scholar
  10. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissues. Focus 12:13–15Google Scholar
  11. Elmowalid GA, Qiao M, Jeong SH, Borg BB, Baumert TF, Sapp RK, Hu ZY, Murthy K, Liang TJ (2007) Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees. Proc Natl Acad Sci USA 104:8427–8432PubMedCrossRefGoogle Scholar
  12. Eugeni Piller L, Abraham M, Dörmann P, Kessler F, Besagni C (2012) Plastid lipid droplets at the crossroads of prenylquinone metabolism. J Ex Bot 63:1609–1618CrossRefGoogle Scholar
  13. Eymery F, Rey P (1999) Immunocytolocalization of CDSP 32 and CDSP 34, two chloroplastic drought-induced stress proteins in Solanum tuberosum plants. Plant Physiol Biochem 37:305–312CrossRefGoogle Scholar
  14. Gaude N, Brehelin C, Tischendorf G, Kessler F, Dormann P (2007) Nitrogen deficiency in Arabidopsis affects galactolipid composition and gene expression and results in accumulation of fatty acid phytyl esters. Plant J 49:729–739PubMedCrossRefGoogle Scholar
  15. Gillet B, Beyly A, Peltier G, Rey P (1998) Molecular characterization of CDSP 34, a chloroplastic protein induced by water deficit in Solanum tuberosum L. plants, and regulation of CDSP 34 expression by ABA and high illumination. Plant J 16:257–262PubMedCrossRefGoogle Scholar
  16. Gisby MF, Mellors P, Madesis P, Ellin M, Laverty H, O’Kane S, Ferguson MWJ, Day A (2011) A synthetic gene increases TGF beta 3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant Biotech J 9:618–628CrossRefGoogle Scholar
  17. Glenz K, Bouchon B, Stehle T, Wallich R, Simon MM, Warzecha H (2006) Production of a recombinant bacterial lipoprotein in higher plant chloroplasts. Nature Biotech 24:76–77CrossRefGoogle Scholar
  18. Humbert N, Zocchi A, Ward TR (2005) Electrophoretic behavior of streptavidin complexed to a biotinylated probe: a functional screening assay for biotin-binding proteins. Electrophoresis 26:47–52PubMedCrossRefGoogle Scholar
  19. Kabeya Y, Nakanishi H, Suzuki K, Ichikawa T, Kondou Y, Matsui M, Miyagishima S (2010) The YlmG protein has a conserved function related to the distribution of nucleoids in chloroplasts and cyanobacteria. BMC Plant Biol 10:57PubMedCrossRefGoogle Scholar
  20. Kessler F, Schnell D, Blobel G (1999) Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts. Planta 208:107–113PubMedCrossRefGoogle Scholar
  21. Kim HU, Wu SSH, Ratnayake C, Huang AHC (2001) Brassica rapa has three genes that encode proteins associated with different neutral lipids in plastids of specific tissues. Plant Physiol 126:330–341PubMedCrossRefGoogle Scholar
  22. Kuroda H, Maliga P (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29:970–975PubMedCrossRefGoogle Scholar
  23. Lichtenthaler HK (1968) Plastoglobuli and fine structure of plastids. Endeavour 27:144–148Google Scholar
  24. Lundquist P, Poliakov A, Bhuiyan NH, Zybailov B, Sun Q, van Wijk KJ (2012) The functional network of the Arabidopsis thaliana plastoglobule proteome based on quantitative proteomics and genome-wide co-expression analysis. Plant Physiol 158:1172–1192PubMedCrossRefGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Obregon P, Chargelegue D, Drake PMW, Prada A, Nuttall J, Frigerio L, Ma JKC (2006) HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production. Plant Biotech 4:195–207CrossRefGoogle Scholar
  27. Pozueta-Romero J, Rafia F, Houlne G, Cheniclet C, Carde JP, Schantz ML, Schantz R (1997) A ubiquitous plant housekeeping gene, PAP, encodes a major protein component of bell pepper chromoplasts. Plant Physiol 115:1185–1194PubMedCrossRefGoogle Scholar
  28. Rey P, Gillet B, Römer S, Eymery F, Massimino J, Peltier G, Kuntz M (2000) Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development under stress. Plant J 21:483–494PubMedCrossRefGoogle Scholar
  29. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotech 19:870–875CrossRefGoogle Scholar
  30. Singh ND, Li M, Lee SB, Schnell D, Daniell H (2008) Arabidopsis Tic40 expression in tobacco chloroplasts results in massive proliferation of the inner envelope membrane and upregulation of associated proteins. Plant Cell 20:3405–3417PubMedCrossRefGoogle Scholar
  31. Smith MD, Hiltbrunner A, Kessler F, Schnell DJ (2002) The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP. J Cell Biol 159:833–843PubMedCrossRefGoogle Scholar
  32. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aada gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  33. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher-plants. Proc Natl Acad Sci USA 87:8526–8530PubMedCrossRefGoogle Scholar
  34. Targett-Adams P, Chambers D, Gledhill S, Hope RG, Coy JF, Girod A, McLauchlan J (2003) Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein. J Biol Chem 278:15998–16007PubMedCrossRefGoogle Scholar
  35. Tissot G, Canard H, Nadai M, Martone A, Botterman J, Dubald M (2008) Translocation of aprotinin, a therapeutic protease inhibitor, into the thylakoid lumen of genetically engineered tobacco chloroplasts. Plant Biotech J 6:309–320CrossRefGoogle Scholar
  36. Tregoning JS, Nixon P, Kuroda H, Svab Z, Clare S, Bowe F, Fairweather N, Ytterberg J, van Wijk KJ, Dougan G, Maliga P (2003) Expression of tetanus toxin Fragment C in tobacco chloroplasts. Nucleic Acids Res 31:1174–1179PubMedCrossRefGoogle Scholar
  37. Vanrooijen GJH, Moloney MM (1995) Plant seed oil-bodies as carriers for foreign proteins. Bio Tech 13:72–77Google Scholar
  38. Vidi PA, Kanwischer M, Baginsky S, Austin JR, Csucs G, Dormann P, Kessler F, Brehelin C (2006) Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles. J Biol Chem 281:11225–11234PubMedCrossRefGoogle Scholar
  39. Vidi PA, Kessler F, Brehelin C (2007) Plastoglobules: a new address for targeting recombinant proteins in the chloroplast. BMC Biotech 7:4CrossRefGoogle Scholar
  40. Wurbs D, Ruf S, Bock R (2007) Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J 49:276–288PubMedCrossRefGoogle Scholar
  41. Ytterberg AJ, Peltier JB, van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes. Plant Physiol 140:984–997PubMedCrossRefGoogle Scholar
  42. Zhou F, Badillo-Corona JA, Karcher D, Gonzalez-Rabade N, Piepenburg K, Borchers A-MI, Maloney AP, Kavanagh TA, Gray JC, Bock R (2008) High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes. Plant Biotech J 6:897–913CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Venkatasalam Shanmugabalaji
    • 3
  • Céline Besagni
    • 1
  • Lucia Eugeni Piller
    • 1
  • Veronique Douet
    • 1
  • Stephanie Ruf
    • 2
  • Ralph Bock
    • 2
  • Felix Kessler
    • 1
  1. 1.Laboratoire de Physiologie VégétaleUniversité de NeuchâtelNeuchâtelSwitzerland
  2. 2.Max-Planck-Institut für Molekulare PflanzenphysiologiePotsdam, GolmGermany
  3. 3.Department of Plant BiologyUniversity of GenevaGeneva 4Switzerland

Personalised recommendations