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Simultaneous Analyses of Oxidized and Reduced Forms of Photosynthetic Quinones by High-Performance Liquid Chromatography

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Plant Isoprenoids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1153))

Abstract

Plastoquinones and phylloquinones are the major plant quinones localized in chloroplasts, and they act as photosynthetic electron redox mediators in thylakoid membranes. These quinones are analyzed by two processes: extraction with organic solvents and quinone assay by high-performance liquid chromatography (HPLC) analysis. Solvent choice is very important from the viewpoint of stability of the redox state in the extraction processes and during storage of plant quinones. We introduce procedures and solvents to avoid changes in the redox state of quinones, in addition to achieving high extraction efficiency. Traditional methods have problems of low sensitivity and require preparation steps to remove interfering substances, such as plant pigments. HPLC systems have been developed utilizing the fluorescent properties of quinols (reduced forms) to measure quinones. Plastoquinones were detected by reversed-phase HPLC with dual detectors (ultra-violet and fluorescence detection). However, the peak of phylloquinone and plastoquinone isomers with shorter side chains often overlaps with a large peak of fast-eluting pigments. To address these issues, HPLC with fluorescence detection after post-column reduction to convert quinones to fluorescent quinol was applied for measurement of fast-eluting quinones (low hydrophobicity quinones and quinols) such as phylloquinone. Using post-column reduction methods with sodium borohydride or platinum black, not only the reduced forms (fluorescent) but also the oxidized forms (non-fluorescent) could be clearly measured by HPLC with a fluorescence detector.

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References

  1. Nowicka B, Kruk J (2010) Occurrence, biosynthesis and function of isoprenoid quinones. Biochim Biophys Acta 1797:1587–1605

    Article  PubMed  CAS  Google Scholar 

  2. Pulido P, Perello C, Rodriguez-Concepcion M (2012) New insights into plant isoprenoid metabolism. Mol Plant 5:964–967

    Article  PubMed  CAS  Google Scholar 

  3. Lichtenthaler HK (2007) Biosynthesis, accumulation and emission of carotenoids, α-tocopherol, plastoquinone, and isoprene in leaves under high photosynthetic irradiance. Photosynth Res 92:163–179

    Article  PubMed  CAS  Google Scholar 

  4. Barr R, Crane FL (1967) Comparative studies on plastoquinones. III. Distribution of plastoquinones in higher plants. Plant Physiol 42:1255–1263

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  5. Okayama S (1984) Reversed-phase high-performance liquid chromatography of prenylquinones in green leaves using an electrochemical detector. Plant Cell Physiol 25:1445–1449

    CAS  Google Scholar 

  6. Barr R, Crane FL (1971) Quinones in algae and higher plants. Methods Enzymol 23:372–408

    Article  CAS  Google Scholar 

  7. Kruk J, Burda K, Schmid GH, Radunz A, Strzalka K (1998) Function of plastoquinones B and C as electron acceptors in photosystem II. Fatty acids analysis of plastoquinone. Photosynth Res 58:203–209

    Article  CAS  Google Scholar 

  8. Lichtenthaler HK, Prenzel U, Douce D, Joyard J (1981) Localization of prenylquinones in the envelope of spinach chloroplasts. Biochim Biophys Acta 641:99–105

    Article  PubMed  CAS  Google Scholar 

  9. Lundquist PK, Poliakov A, Bhuiyan NH, Zybailov B, Sun Q, van Wijk KJ (2012) The functional network of the arabidopsis plastoglobule proteome based on quantitative proteomics and genome-wide coexpression analysis. Plant Physiol 158:117–1192

    Google Scholar 

  10. Kruk J, Trebst A (2008) Plastoquinol as a singlet oxygen scavenger in photosystem II. Biochim Biophys Acta 1777:154–162

    Article  PubMed  CAS  Google Scholar 

  11. Nowicka B, Kruk J (2012) Plastoquinol is more active than α-tocopherol in singlet oxygen scavenging during high light stress of Chlamydomonas reinhardtii. Biochim Biophys Acta 1817:389–394

    Article  PubMed  CAS  Google Scholar 

  12. Pshybytko NL, Kruk J, Kabashnikova LF, Strzalka K (2008) Function of plastoquinone in heat stress reactions of plants. Biochem Biophys Acta 1777:1393–1399

    PubMed  CAS  Google Scholar 

  13. Zer H, Ohad I (2003) Light, redox state, thylakoid-protein phosphorylation and signaling gene expression. Trends Biochem Sci 28:467–470

    Article  PubMed  CAS  Google Scholar 

  14. Johnson TW, Zybailov B, Jones AD, Bittl R, Zech S, Stehlik D, Golbeck JH, Chitnis PR (2001) Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803. J Biol Chem 276:39512–39521

    Article  PubMed  CAS  Google Scholar 

  15. Shimada H, Ohno R, Shibata M, Ikegami I, Onai K, Ohto M, Takamiya K (2005) Inactivation and deficiency of core proteins of photosystems I and II caused by genetical phylloquinone and plastoquinone deficiency but retained lamellar structure in a T-DNA mutant of Arabidopsis. Plant J 41:627–637

    Article  PubMed  CAS  Google Scholar 

  16. Kruk J, Karpinski S (2006) An HPLC-based method of estimation of the total redox state of plastoquinone in chloroplasts, the size of the photochemically active plastoquinone-pool and its redox state in thylakoids of Arabidopsis. Biochem Biophys Acta 1757:1669–1675

    PubMed  CAS  Google Scholar 

  17. Yoshida K, Shibata M, Terashima I, Noguchi K (2010) Simultaneous determination of in vivo plastoquinone and ubiquinone redox states by HPLC-based analysis. Plant Cell Physiol 51:836–841

    Article  PubMed  CAS  Google Scholar 

  18. Martinis J, Kessler F, Glauser G (2011) A novel method for prenylquinone profiling in plant tissues by ultra-high pressure liquid chromatography-mass spectrometry. Plant Methods 7:23, doi: 2310.1186/1746-4811-7-23

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  19. Isogai Y, Mishimura M, Okayama S (1987) Simultaneous determination of oxidized and reduced forms of plastoquinone A in spinach chloroplasts by high-performance liquid chromatography with an electrochemical detector. Plant Cell Physiol 28:1301–1306

    CAS  Google Scholar 

  20. Yoshida K, Watababe CK, Hachiya T, Tholen D, Shibata M, Terashima I, Noguchi K (2011) Distinct responses of the mitochondrial respiratory chain to long- and short-term high-light environments in Arabidopsis thaliana. Plant Cell Physiol 34:618–628

    CAS  Google Scholar 

  21. Cheng Z, Sattler S, Maeda H, Sakuragi Y, Bryant DA, Penna DD (2003) Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in cyanobacteria and photosynthetic eukaryotes. Plant Cell 15:2343–2356

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  22. Hiraike H, Kimura M, Itokawa Y (1986) Measurement of K vitamins in human placenta by high-performance liquid chromatography with fluorometric detection. Jpn J Hyg 41:764–768

    Article  CAS  Google Scholar 

  23. Hiraike H, Kimura M, Itokawa Y (1988) Determination of K vitamins (phylloquinone and menaquinones) in umbilical cord plasma by a platinum-reduction column. J Chromatogr 430:143–148

    Article  PubMed  CAS  Google Scholar 

  24. Lohmann A, Schöttler M, Brėhėlin C, Kessler F, Bock R, Cahoon EB, Doėrmann P (2006) Deficiency in phylloquinone (Vitamin K1) methylation affects prenyl quinone distribution, photosystem I abundance, and anthocyanin accumulation in the Arabidopsis AtmenG mutant. J Biol Chem 281:40461–40472

    Article  PubMed  CAS  Google Scholar 

  25. Jakob E, Elmadfa I (1996) Application of a simplified HPLC assay for the determination of phylloquinone (vitamin K1) in animal and plant food items. Food Chem 56:87–91

    Article  CAS  Google Scholar 

  26. Shibata M, Tsuyama M, Takami T, Shimizu H, Kobayashi Y (2004) Accumulation of menaquinones with incompletely reduced side chains and loss of α-tocopherol in rice mutants with alternations in the chlorophyll moiety. J Exp Bot 55:1989–1996

    Article  PubMed  CAS  Google Scholar 

  27. Kusube K, Abe K, Hiroshima O, Ishiguro Y, Ishikawa S, Hoshida H (1984) Determination of vitamin K analogues by high performance liquid chromatography with electrochemical derivatization. Chem Pharm Bull 32:179–184

    Article  PubMed  CAS  Google Scholar 

  28. De Vitry C, Carles C, Diner BA (1986) Quantitation of plastquinone-9 in photosystem II reaction center particles. FEBS Lett 196:203–206

    Article  Google Scholar 

  29. Kruk J, Strzalka K, Leblanc RM (1993) Fluorescence properties of plastoquinol, ubiquinol and α-tocopherol quinol in solution and liposome membranes. J Photochem Photobiol B Biol 19:33–38

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Faculty of Education, Biological Institute, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, Japan

    Masaru Shibata

  2. Department of Mathematical and Life Sciences, Laboratory of Molecular Plant Biology, Graduate School of Sciences, Hiroshima University, 1-3-1, Kagamiyama, Higashi-Hiroshima, Japan

    Hiroshi Shimada

Authors
  1. Masaru Shibata
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  2. Hiroshi Shimada
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Correspondence to Hiroshi Shimada .

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Editors and Affiliations

  1. Centre for Research in Agricultural Genomic (CRAG), CSIC-IRTA-UAB-UB, Barcelona, Spain

    Manuel Rodríguez-Concepción

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Shibata, M., Shimada, H. (2014). Simultaneous Analyses of Oxidized and Reduced Forms of Photosynthetic Quinones by High-Performance Liquid Chromatography. In: Rodríguez-Concepción, M. (eds) Plant Isoprenoids. Methods in Molecular Biology, vol 1153. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0606-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-0606-2_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0605-5

  • Online ISBN: 978-1-4939-0606-2

  • eBook Packages: Springer Protocols

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