Photosynthesis Research

, Volume 74, Issue 2, pp 109–119 | Cite as

Chlorophyll breakdown in spinach: on the structure of five nonfluorescent chlorophyll catabolites*

  • Joachim Berghold
  • Kathrin Breuker
  • Michael Oberhuber
  • Stefan Hörtensteiner
  • Bernhard Kräutler

Abstract

In extracts of senescent leaves of spinach (Spinacia oleracea), five colourless compounds with UV/Vis-characteristics of nonfluorescent chlorophyll catabolites (NCCs) were detected and tentatively named So-NCCs. The most abundant polar NCC in the leaves of this vegetable, So-NCC-2, had been isolated earlier and its constitution was determined by spectroscopic means. The catabolite So-NCC-2 was found to be an epimer of a polar NCC from barley (Hordeum vulgare), the first non-green chlorophyll catabolite from a higher plant to be structurally analyzed. Here, we report on the isolation of four additional So-NCCs from the extracts of senescent leaves of Sp. oleracea by two- (or multi-)stage chromatographic purification and on their structural characterization. The constitution of So-NCC-3 could be determined by spectroscopic analysis in combination with chemical correlation with a known NCC from Cercidiphyllum japonicum (Cj-NCC): So-NCC-3 was identified as the hydrolysis product of the methyl ester function of Cj-NCC. The less polar catabolite So-NCC-4 could be directly identified with Cj-NCC. Two further So-NCCs, So-NCC-1 and So-NCC-5, were detected only in trace amounts. Five structurally related nonfluorescent chlorophyll catabolites (So-NCCs) are thus present in senescent leaves of spinach. The structures of this set of So-NCCs indicate several peripheral refunctionalization reactions and inform on the late catabolic transformations during leaf senescence. The transformation of the tetrapyrrolic skeleton in chlorophyll catabolism in spinach and in C. japonicum is revealed to exhibit a common stereochemical pattern.

chlorophyll chlorophyll catabolism mass spectrometry NMR-spectroscopy partial synthesis tetrapyrrole 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bortlik K-H, Peisker C and Matile P (1990) A novel type of chlorophyll catabolite in senescent barley leaves. J Plant Physiol 136: 161–165Google Scholar
  2. Brown SB, Houghton JD and Hendry GAF (1991) Chlorophyll breakdown. In:Scheer H (ed) Chlorophylls, pp 465–489. CRC Press, Boca Raton, FloridaGoogle Scholar
  3. Curty C and Engel N (1996) Detection, isolation and structure elucidation of a chlorophyll a catabolite from autumnal senescent leaves of Cercidiphyllum japonicum. Phytochemistry 42: 1531–1536CrossRefGoogle Scholar
  4. Doi M, Inage T and Shioi Y (2001) Chlorophyll degradation in a Chlamydomonas reinhardtii mutant: an accumulation of pyropheophorbide a by anaerobiosis. Plant Cell Physiol 42: 469–474PubMedCrossRefGoogle Scholar
  5. Ginsburg S and Matile P (1993) Identification of catabolites of chlorophyll porphyrin in senescent rape cotyledons. Plant Physiol 102: 521–527PubMedGoogle Scholar
  6. Hendry GAF, Houghton JD and Brown SB (1987) The degradation of chlorophyll-a biological enigma. New Phytol 107: 255–302CrossRefGoogle Scholar
  7. Hinder B, Schellenberg M, Rodoni S, Ginsburg S, Vogt E, Martinoia E, Matile P and Hörtensteiner S (1996) How plants dispose of chlorophyll catabolites. Directly energized uptake of tetrapyrrolic breakdown products into isolated vacuoles. J Biol Chem 271: 27233–27236PubMedCrossRefGoogle Scholar
  8. Hörtensteiner S (1998a) NCC malonyltransferase catalyses the fi-nal step of chlorophyll breakdown in rape (Brassica napus). Phytochemistry 49: 953–956PubMedCrossRefGoogle Scholar
  9. Hörtensteiner S and Kräutler B (2000) Chlorophyll breakdown in oilseed rape. Photosynth Res 64: 137–146PubMedCrossRefGoogle Scholar
  10. Hörtensteiner S, Vicentini F and Matile P (1995) Chlorophyll breakdown in senescent cotyledons of rape, Brassica napus L.: enzymatic cleavage of phaeophorbide a in vitro. New Phytol 129: 237–246CrossRefGoogle Scholar
  11. Hörtensteiner S, Wüthrich KL, Matile P, Ongania K-H and Kräutler B (1998b) The key step in chlorophyll breakdown in higher plants. Cleavage of pheophorbide a macrocycle by a monooxygenase. J Biol Chem 273: 15335–15339PubMedCrossRefGoogle Scholar
  12. Hörtensteiner S, Rodoni S, Schellenberg M, Vicentini F, Nandi OI, Qiu Y-L and Matile P (2000) Evolution of chlorophyll degradation: the significance of RCC reductase. Plant Biol 2: 63–67CrossRefGoogle Scholar
  13. Ito H, Tanaka Y, Tsuji H and Tanaka A (1993) Conversion of chlorophyll b to chlorophyll a by isolated cucumber etioplasts. Arch Biochem Biophys 306: 148–151PubMedCrossRefGoogle Scholar
  14. Ito H, Ohysuka T and Tanaka A (1996) Conversion of chlorophyll b to chlorophyll a via 7-hydroxymethyl chlorophyll. J Biol Chem 271: 1475–1479PubMedCrossRefGoogle Scholar
  15. Iturraspe J, Moyano N and Frydman B (1995) A new 5-formylbilinone as the major chlorophyll a catabolite in tree senescent leaves. J Org Chem 60: 6664–6665CrossRefGoogle Scholar
  16. Kessler H, Gehrke M and Griesinger C (1988) Zweidimensionale NMR-Spektroskopie, Grundlagen und Ñbersicht über die Experimente. Angew Chem 100: 507–544; Angew Chem Int Ed Engl 27: 490-537Google Scholar
  17. Kräutler B and Matile P (1999) Solving the riddle of chlorophyll breakdown. Acc Chem Res 32: 35–43CrossRefGoogle Scholar
  18. Kräutler B, Jaun B, Bortlik K-H, Schellenberg M and Matile P (1991) On the enigma of chlorophyll degradation: the constitution of a secoporphinoid catabolite. Angew Chem Int Ed Engl 30: 1315–1318CrossRefGoogle Scholar
  19. Kräutler B, Jaun B, Amrein W, Bortlik K, Schellenberg M and Matile P (1992) Breakdown of chlorophyll: constitution of a secoporphinoid chlorophyll catabolite isolated from senescent barley leaves. Plant Physiol Biochem 30: 333–346Google Scholar
  20. Losey FG and Engel N (2001) Isolation and characterization of a urobilinogenoidic chlorophyll catabolite from Hordeum vulgare. J Biol Chem 276: 8643–8647PubMedCrossRefGoogle Scholar
  21. Lu Y-P, Li Z-S, Drozdowicz Y-M, Hörtensteiner S, Martinoia E and Rea PA (1998) AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: functional comparisons with AtMRP1. Plant Cell 10: 267–282PubMedCrossRefGoogle Scholar
  22. Matile P (1987) Seneszenz bei Pflanzen und ihre Bedeutung für den Stickstoffhaushalt. Chimia 41: 376–381Google Scholar
  23. Matile P and Kräutler B (1995) Wie und warum bauen Pflanzen das Chlorophyll ab. Chem unserer Zeit 29: 298–306CrossRefGoogle Scholar
  24. Matile P, Ginsburg S, Schellenberg M and Thomas H (1988) Catabolites of chlorophyll in senescing barley leaves are localized in the vacuoles of mesophyll cells. Proc Natl Acad Sci USA 85: 9529–9532PubMedCrossRefGoogle Scholar
  25. Matile P, Hörtensteiner S, Thomas H and Kräutler B (1996) Chlorophyll breakdown in senescent leaves. Plant Physiol 112: 1403–1409PubMedGoogle Scholar
  26. Mendel G (1865) Versuche über Pflanzenhybriden. Verhandlungen des Naturwissenschaftlichen Vereins, Brünn 4: 3–47Google Scholar
  27. Mühlecker W and Kräutler B (1996) Breakdown of chlorophyll: constitution of nonfluorescing chlorophyll-catabolites from senescent cotyledons of the dicot rape. Plant Physiol Biochem 34: 61–75Google Scholar
  28. Mühlecker W, Kräutler B, Ginsburg S and Matile P (1993) Breakdown of chlorophyll: the constitution of a secoporphinoid chlorophyll catabolite from senescent rape leaves. Helv Chim Acta 76: 2976–2980CrossRefGoogle Scholar
  29. Mühlecker W, Ongania K-H, Kräutler B, Matile P and Hörtensteiner S (1997) Tracking down chlorophyll breakdown in plants: elucidation of the constitution of a fluorescent chlorophyll catabolite. Angew Chem Int Ed Engl 36: 401–404CrossRefGoogle Scholar
  30. Mühlecker W, Kräutler B, Moser D, Matile P and Hörtensteiner S (2000) Breakdown of chlorophyll: a fluorescent chlorophyll catabolite from sweet pepper (Capsicum annuum). Helv Chim Acta 83: 278–286CrossRefGoogle Scholar
  31. Oberhuber M, Berghold J, Mühlecker W, Hörtensteiner S and Kräutler B (2001) Chlorophyll breakdown-on a nonfluorescent chlorophyll catabolite from spinach. Helv Chim Acta 84: 2615–2627CrossRefGoogle Scholar
  32. Pretsch E, Bühlmann P and Affolter C (2000) Structure Determination of Organic Compounds, pp 158–160. Springer-Verlag, BerlinGoogle Scholar
  33. Rodoni S, Vicentini F, Schellenberg M, Matile P and Hörtensteiner S (1997) Partial purification and characterization of red chlorophyll catabolite reductase, a stroma protein involved in chlorophyll breakdown. Plant Physiol 115: 677–682PubMedCrossRefGoogle Scholar
  34. Sanders JKM and Hunter BK (1987) Modern NMR-spectroscopy. Oxford University Press, Oxford Scheer H (ed) (1991) Chlorophylls. CRC-Press, Boca Raton, FloridaGoogle Scholar
  35. Scheumann V, Ito H, Tanaka A, Schoch S and Rüdiger W (1996) Substrate specificity of chlorophyll(ide) b reductase in etioplasts of barley (Hordeum vulgare). Eur J Biochem 242: 163–170PubMedCrossRefGoogle Scholar
  36. Scheumann V, Schoch S and Rüdiger W (1999) Chlorophyll b reduction during senescence of barley seedlings. Planta 209: 364–370PubMedCrossRefGoogle Scholar
  37. Shioi Y, Watanabe K and Takamiya K (1996) Enzymatic conversion of pheophorbide a to a precursor of pyropheophorbide a in leaves of Chenopodium album. Plant Cell Physiol 37: 1143–1149Google Scholar
  38. Smith RD, Light-Wahl KJ, Winger BE and Goodlett DR (1995) Electrospray Ionization. In: Matsuo T, Caprioli RM, Gross ML and Seyama Y (eds) Biological Mass Spectrometry: Present and Future, pp 41–74. John Wiley & Sons, Chichester, UKGoogle Scholar
  39. Tommasini R, Vogt E, Fromenteau M, Hörtensteiner S, Matile P, Amrhein N and Martinoia E (1998) An ABC transporter of Arabidopsis thaliana has both glutathione-conjugate and chlorophyll catabolite transport activity. Plant J 13: 773–780PubMedCrossRefGoogle Scholar
  40. Watson TR (1995) Fast atom bombardment. In: Matsuo T, Caprioli RM, Gross ML and Seyama Y (eds) Biological Mass Spectrometry: Present and Future, pp 24–40. John Wiley & Sons, Chichester, UKGoogle Scholar
  41. Wüthrich KL, Bovet L, Hunziker PE, Donnison IS and Hörtensteiner S (2000) Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism. Plant J 21: 189–198PubMedCrossRefGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Joachim Berghold
    • 1
  • Kathrin Breuker
    • 1
  • Michael Oberhuber
    • 1
  • Stefan Hörtensteiner
    • 2
  • Bernhard Kräutler
    • 1
  1. 1.Institute of Organic ChemistryLeopold-Franzens-Universität InnsbruckInnsbruckAustria
  2. 2.Institute of Plant SciencesUniversität BernBernSwitzerland

Personalised recommendations