Skip to main content

Advertisement

SpringerLink
Log in

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

BSA:

Bovine serum albumin

cTP:

Chloroplast transit peptide

FT:

Fourier transform

ICR:

Ion cyclotron resonance

LTQ:

Linear quadruple trap

PIR:

Protein information resources

SP:

Signal peptide

TMD:

Transmembrane domain

References

  1. Bryant DA, Frigaard NU (2006) Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol 14:488–496

    Article  PubMed  CAS  Google Scholar 

  2. Sheen J (1999) C-4 gene expression. Annu Rev Plant Physiol Plant Mol Biol 50:187–217

    Article  PubMed  CAS  Google Scholar 

  3. Ort DR, Yocum CF (1996) Electron transfer and energy transduction in photosynthesis: an overview. In: Ort DR, Yocum CF (eds) Oxygenic photosynthesis: the light reactions. Advances in photosynthesis and respiration. Springer, Dordrecht, pp 1–9

    Google Scholar 

  4. van Wijk KJ (2004) Plastid proteomics. Plant Physiol Biochem 42:963–977

    Article  PubMed  Google Scholar 

  5. Jung E, Heller M, Sanchez JC, Hochstrasser DF (2000) Proteomics meets cell biology: the establishment of subcellular proteomes. Electrophoresis 21:3369–3377

    Article  PubMed  CAS  Google Scholar 

  6. van Wijk KJ (2001) Challenges and prospects of plant proteomics. Plant Physiol 126:501–508

    Article  PubMed  Google Scholar 

  7. Bardel J, Louwagie M, Jaquinod M et al (2000) A survey of the plant mitochondrial proteome in relation to development. Proteomics 2:880–898

    Article  Google Scholar 

  8. Millar AH, Sweetlove LJ, Giege P, Leaver CJ (2001) Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127:1711–1727

    Article  PubMed  CAS  Google Scholar 

  9. van Wijk KJ (2000) Proteomics of the chloroplast: experimentation and prediction. Trends Plant Sci 5:420–425

    Article  Google Scholar 

  10. Friso G, Giacomelli L, Ytterberg AJ, Peltier JB et al (2004) In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database. Plant Cell 16:478–499

    Article  PubMed  CAS  Google Scholar 

  11. Peltier JB, Ytterberg AJ, Sun Q, van Wijk KJ (2004) New functions of the thylakoid membrane proteome of Arabidopsis thaliana revealed by a simple, fast, and versatile fractionation strategy. J Biol Chem 279:49367–49383

    Article  PubMed  CAS  Google Scholar 

  12. Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T (2000) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329–3344

    Article  PubMed  CAS  Google Scholar 

  13. Marmagne A, Rouet MA, Ferro M, Rolland N et al (2004) Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome. Mol Cell Proteomics 3:675–691

    Article  PubMed  CAS  Google Scholar 

  14. Mitra SK, Gantt JA, Ruby JF, Clouse SD, Goshe MB (2007) Membrane proteomic analysis of Arabidopsis thaliana using alternative solubilization techniques. J Proteome Res 6:1933–1950

    Article  PubMed  CAS  Google Scholar 

  15. Fukao Y, Hayashi M, Nishimura M (2002) Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant Cell Physiol 43:689–696

    Article  PubMed  CAS  Google Scholar 

  16. Maltman DJ, Simon WJ, Wheeler CH, Dunn MJ et al (2002) Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis). Electrophoresis 23:626–629

    Article  PubMed  CAS  Google Scholar 

  17. Chivasa S, Ndimba BK, Simon WJ, Robertson D (2002) Proteomic analysis of the Arabidopsis thaliana cell wall. Electrophoresis 23:1754–1765

    Article  PubMed  CAS  Google Scholar 

  18. Degenhardt RF, Bonham-Smith PC (2008) Arabidopsis ribosomal proteins RPL23aA and RPL23aB are differentially targeted to the nucleolus and are disparately required for normal development. Plant Physiol 147:128–142

    Article  PubMed  CAS  Google Scholar 

  19. Ferro M, Salvi D, Brugière S, Miras S, Kowalski S et al (2003) Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana. Mol Cell Proteomics 2:325–345

    PubMed  CAS  Google Scholar 

  20. Peltier JB, Friso G, Kalume DE, Roepstorff P et al (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12:319–342

    Article  PubMed  CAS  Google Scholar 

  21. Schubert M, Petersson UA, Haas BJ, Funk C et al (2000) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J Biol Chem 277:8354–8365

    Article  Google Scholar 

  22. Kiselbach T, Hagman A, Anderson B, Schroder WP (1998) The thylakoid lumen of chloroplasts. J Biol Chem 20:6710–6716

    Article  Google Scholar 

  23. D’Amici GM, Huber GC, Zolla L (2009) Separation of thylakoid membrane proteins by sucrose gradient ultracentrifuge or blue native-SDS-PAGE two-dimensional electrophoresis. In: Peirce MJ, Waits R (eds) Membrane proteomics: methods and protocols. Springer, New York, pp 61–70

  24. Hippler M, Klein J, Fink A, Allinger T, Hoerth P (2001) Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinharditii. Plant J 28:595–606

    Article  PubMed  CAS  Google Scholar 

  25. Kikuchi S, Hirohashi T, Nakai M (2006) Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by Blue Native PAGE. Plant Cell Physiol 47:363–371

    Article  PubMed  CAS  Google Scholar 

  26. Sugiyama N, Nakagami H, Mochida K, Daudi A (2008) Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis. Mol Syst Biol 4:193

    Article  PubMed  Google Scholar 

  27. Krijgsveld J, Gauci S, Dormeyer W, Heck AJ (2006) In-gel isoelectric focusing of peptides as a tool for improved protein identification. J Proteome Res 5:1721–1730

    Article  PubMed  CAS  Google Scholar 

  28. Cao X, Nesvizhskii AI (2008) Improved sequence tag generation method for peptide identification in tandem mass spectrometry. J Proteome Res 7:4422–4434

    Article  PubMed  CAS  Google Scholar 

  29. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents; verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim et Biophys Acta 975:384–394

    Article  CAS  Google Scholar 

  30. Zorb C, Herbst R, Forreiter C, Schubert S (2009) Short-term effects of salt exposure on the maize chloroplast protein pattern. Proteomics 9:4209–4220

    Article  PubMed  Google Scholar 

  31. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

    Article  PubMed  CAS  Google Scholar 

  32. Kim JY, Lee JH, Park GW, Cho K, Kwon KH et al (2005) Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis. Proteomics 5:3376–3385

    Article  PubMed  CAS  Google Scholar 

  33. Olsen JV, Mann M (2004) Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. Proc Natl Acad Sci USA 101:13417–13422

    Article  PubMed  CAS  Google Scholar 

  34. Horton P, Park KJ, Obayashi T, Fujita N et al (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35:585–587

    Article  Google Scholar 

  35. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36

    Article  PubMed  CAS  Google Scholar 

  36. Emanuelsson O, Nielsen H, von Heijne G (1999) ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci 8:978–984

    Article  PubMed  CAS  Google Scholar 

  37. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

    Article  PubMed  CAS  Google Scholar 

  38. Möller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653

    Article  PubMed  Google Scholar 

  39. Ishihama Y, Oda Y, Tabata T, Sato T et al (2005) Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol Cell Proteomics 4:1265–1272

    Article  PubMed  CAS  Google Scholar 

  40. Matsuoka M (1990) Classification and characterization of cDNA that encodes the light-harvesting chlorophyll a/b binding protein of photosystem II from rice. Plant Cell Physiol 31:519–526

    CAS  Google Scholar 

  41. Lemieux C, Otis C, Turmel M (2000) Ancestral chloroplast genome in Mesostigma viride reveals an early branch of green plant evolution. Nature 403:649–652

    Article  PubMed  CAS  Google Scholar 

  42. Yuan HM, Li KL, Ni RJ, Guo WD et al (2010) A systemic proteomic analysis of Populus chloroplast by using shotgun method. Mol Biol Rep. doi:10.1007/s11033-010-9971-y

  43. Ferro M, Salvi D, Rivière-Rolland H, Vermat T et al (2002) Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters. Proc Natl Acad Sci USA 99:11487–11492

    Article  PubMed  CAS  Google Scholar 

  44. Syka JE, Marto JA, Bai DL, Horning S et al (2004) Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications. J Proteome Res 3:326–621

    Article  Google Scholar 

  45. Peltier JB, Emanuelsson O, Kalume DE, Ytterberg J et al (2002) Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14:211–236

    Article  PubMed  CAS  Google Scholar 

  46. Gómez SM, Bil KY, Aguilera R, Nishio JN et al (2003) Transit peptide cleavage sites of integral thylakoid membrane proteins. Mol Cell Proteomics 2:1068–1085

    Article  PubMed  Google Scholar 

  47. Schwartz R, Ting CS, King J (2001) Whole proteome pi values correlate with subcellular localizations of proteins for organisms within the three domains of life. Genome Res 11:703–709

    Article  PubMed  CAS  Google Scholar 

  48. Dalbey RE, Robinson C (1999) Protein translocation into and across the bacterial plasma membrane and the plant thylakoid membrane. Trends Biochem Sci 24:17–22

    Article  PubMed  CAS  Google Scholar 

  49. Keegstra K, Cline K (1999) Protein import and routing systems of chloroplasts. Plant Cell 11:557–570

    Article  PubMed  CAS  Google Scholar 

  50. Cristóbal S, de Gier JW, Nielsen H, von Heijne G (1999) Competition between Sec- and TAT-dependent protein translocation in Escherichia coli. EMBO J 18:2982–2990

    Article  PubMed  Google Scholar 

  51. Claros MG, von Heijne G (1994) TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686

    PubMed  CAS  Google Scholar 

  52. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580

    Article  PubMed  CAS  Google Scholar 

  53. Tanford C (1978) Hydrophobic effect and the organization of living matter. Science 200:1012–1018

    Article  PubMed  CAS  Google Scholar 

  54. Kruger NJ, von Schaewen A (2003) The oxidative pentose phosphate pathway: structure and organization. Curr Opin Plant Biol 6:236–246

    Article  PubMed  CAS  Google Scholar 

  55. Wall MK, Mitchenall LA, Maxwell A (2004) Arabidopsis thaliana DNA gyrase is targeted to chloroplasts and mitochondria. Proc Natl Acad Sci USA 101:7821–7826

    Article  PubMed  CAS  Google Scholar 

  56. Dupont FM (2008) Metabolic pathways of the wheat (Triticum aestivum) endosperm amyloplast revealed by proteomics. BMC Plant Biol 8:39

    Article  PubMed  Google Scholar 

  57. Huang H, Barker WC, Chen Y, Wu CH (2003) iProClass: an integrated database of protein family, function and structure information. Nucleic Acids Res 31:390–392

    Article  PubMed  CAS  Google Scholar 

  58. Martin W, Herrmann RG (1998) Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol 118:9–17

    Article  PubMed  CAS  Google Scholar 

  59. Ogihara Y, Isono K, Kojima T, Endo A et al (2000) Chinese spring wheat (Triticum aestivum L.) chloroplast genome: complete sequence and contig clones. Plant Mol Biol Rep 18:243–253

    Article  CAS  Google Scholar 

  60. Tang J, Xia H, Cao M, Zhang X et al (2004) A comparison of rice chloroplast genomes. Plant Physiol 135:412–420

    Article  PubMed  CAS  Google Scholar 

  61. Mallick P, Schirle M, Chen SS, Flory MR et al (2007) Computational prediction of proteotypic peptides for quantitative proteomics. Nat Biotechnol 25:125–131

    Article  PubMed  CAS  Google Scholar 

  62. MacKintosh C (2004) Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes. Biochem J 381:329–342

    Article  PubMed  CAS  Google Scholar 

  63. Li J (2005) Brassinosteroid signaling: from receptor kinases to transcription factors. Curr Opin Plant Biol 8:526–531

    Article  PubMed  CAS  Google Scholar 

  64. Wu K, Rooney MF, Ferl RJ (1997) The Arabidopsis 14-3-3 multigene family. Plant Physiol 114:1421–1431

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Prof. Shien Young Kang of the Department of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National University, Korea, for conducting the ultra-centrifugation. Mainly, financial support for this study was obtained from the AGENDA (20090101036022), RDA, Korea to S. H. Woo, and also technically and financially supported by the Korea Basic Science Institute Grant (G30121) to Kun Cho and Korea Basic Science Institute K-MeP Project (T30110) to J.-S. Choi.

Author information

Authors and Affiliations

  1. Department of Crop Science, Chungbuk National University, 410 Seongbong-ro, Heungdeok-gu, Cheongju, Chungbuk, 361-763, Korea

    Abu Hena Mostafa Kamal & Sun Hee Woo

  2. Mass Spectrometry Research Center, Korea Basic Science Institute, Chungbuk, 863-883, Korea

    Kun Cho

  3. National Institute of Crop Science, NARO, Tsukuba, 305-8517, Japan

    Setsuko Komatsu

  4. Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, 980-8579, Japan

    Nobuyuki Uozumi

  5. Division of Life Science, Korea Basic Science Institute, Daejeon, 305-333, Korea

    Jong-Soon Choi

  6. Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, 305-764, Korea

    Jong-Soon Choi

Authors
  1. Abu Hena Mostafa Kamal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kun Cho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Setsuko Komatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nobuyuki Uozumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jong-Soon Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sun Hee Woo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sun Hee Woo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11033_2011_1302_MOESM1_ESM.xls

Supplementary Table 1 Detailed proteomic data for proteins detected in the chloroplast fraction. The table provides the protein accession number (UniProt_Sprot database), protein descriptions, taxonomy, gene name, subcellular location predicted by WoLF PSORT and PSORT, protein score, molecular weight (MW), protein length, protein matches, peptide queries, protein coverage, iso-electric point (pI), exponential modified protein abundance index (emPAI), chloroplast transit peptide (cTP), signal peptide (SP), transmembrane domains (TMD), and gene ontology-based functional classifications according to biological process using the iProClass database. (XLS 268 kb)

Supplementary Table 2 List of other sub-cellular proteins detected in the chloroplast fractions. (DOC 413 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kamal, A.H.M., Cho, K., Komatsu, S. et al. Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome. Mol Biol Rep 39, 5069–5083 (2012). https://doi.org/10.1007/s11033-011-1302-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-011-1302-4

Keywords

Search

Navigation