Journal of Plant Biology

, Volume 62, Issue 1, pp 27–38 | Cite as

Chlorophyll Degradation and Light-harvesting Complex II Aggregate Formation During Dark-induced Leaf Senescence in Arabidopsis Pheophytinase Mutants

  • Young Nam Yang
  • Rana B. Safarova
  • So-Yon Park
  • Yasuhito Sakuraba
  • Min-Hyuk Oh
  • Ismayil S. Zulfugarov
  • Chin Bum Lee
  • Ayumi Tanaka
  • Nam-Chon PaekEmail author
  • Choon-Hwan LeeEmail author
Original Article


The stay-green mutant of Arabidopsis thaliana, ore10 forms stable light-harvesting complex II (LHCII) aggregates during dark-induced senescence, which showed a single base deletion (G1351) in the coding region of the pheophytinase (PPH) gene. PPH specifically dephytylates the Mg-free chlorophyll (Chl) pigment pheophytin, yielding pheophorbide. In both ore10 and pph-1 mutants, pheophytin a accumulated due to the deficiency of PPH gene, but the amount was relatively smaller than that of degraded Chl, and most of the pheophytin a was bound to the stable LHCII forming aggregates with some other Chl-protein (CP) complexes. Comparison of Chl a/b ratios in thylakoids, aggregates, and LHCII indicated that the suppression of Chl b to Chl a conversion was stronger when Chl b reductase was missing and weak when PPH is missing in the large Chl catabolic complex, which allowed the partial degradation of Chl b. These results suggest that the PPH-dependent pathway is not specific for LHCII, but common for all CP complexes, including LHCII. In PPH-deficient mutants, the degradation of LHCII was suppressed by the formation of aggregates, and some of the remaining CP complexes and pheophytin a were included in the aggregates. Non-included CP complexes were degraded via an unknown mechanism.


Aggregate Chlorophyll degradation Chlorophyllprotein complexes Dark-induced senescence Light-harvesting 


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Supplementary material

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  1. Allen KD, Staehelin LA (1991) Resolution of 16 to 20 chlorophyllprotein complexes using a low ionic strength native green gel system. Anal Biochem 194:214–222CrossRefGoogle Scholar
  2. Aro EM, Virgin I, Andersson B (1993) Photoinhibition of photosystem II. Inactivation, protein damage and turnover. Biochim Biophys Acta 1143:113–134CrossRefGoogle Scholar
  3. Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469CrossRefGoogle Scholar
  4. Hinder B, Schellenberg M, Rodoni S, Ginsburg S, Vogt E, Martinoia E, Matile P, Hörtensteiner S (1996) How plants dispose of chlorophyll catabolites. Directly energized uptake of tetrapyrrolic breakdown products into isolated vacuoles. J Biol Chem 271:27233–27236Google Scholar
  5. Horie Y, Ito H, Kusaba M, Tanaka R, Tanaka A (2009) Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b-protein complexes in Arabidopsis. J Biol Chem 284:17449–17456CrossRefGoogle Scholar
  6. Hörtensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77CrossRefGoogle Scholar
  7. Hörtensteiner S, Kräutler B (2011) Chlorophyll breakdown in higher plants. Biochim Biophys Acta 1807:977–988CrossRefGoogle Scholar
  8. Ito H, Tanaka Y, Tsuji H, Tanaka A (1993) Conversion of chlorophyll b to chlorophyll a by isolated cucumber etioplasts. Arch Biochem Biophys 306:148–151CrossRefGoogle Scholar
  9. Jacob-Wilk D, Holland D, Goldschmidt EE, Riov J, Eyal Y (1999) Chlorophyll breakdown by chlorophyllase:isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development. Plant J 20:653–661CrossRefGoogle Scholar
  10. Jeffrey SW, Mantoura RFC, Wright SW (1997) Pheophytin a. In SW Jeffrey, RFC Mantoura, SW Wright, eds, Phytoplankton pigments in oceanography:guidelines to modern methods. UNESCO, Paris, pp462−463Google Scholar
  11. Konieczny A, Ausubel FM (1993) A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers. Plant J 4:403–410CrossRefGoogle Scholar
  12. Kräutler B, Jaun B, Bortlik K, Schellenberg M, Matile P (1991) On the enigma of chlorophyll degradation:the constitution of a secoporphinoid catabolite. Angew Chem Int Ed Engl 30:1315–1318CrossRefGoogle Scholar
  13. Kräutler B (2008) Chlorophyll breakdown and chlorophyll catabolites in leaves and fruit. Photochem Photobiol Sci 7:1114–1120CrossRefGoogle Scholar
  14. Kusaba M, Ito H, Morita R, Iida S, Sato Y, Fujimoto M, Kawasaki S, Tanaka R, Hirochika H, Nishimura M, Tanaka A (2007) Rice NON-YELLOW COLORING1 is involved in light-harvesting complex II and grana degradation during leaf senescence. Plant Cell 19:1362–1375CrossRefGoogle Scholar
  15. Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X, Chang W (2004) Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428:287–292CrossRefGoogle Scholar
  16. Matile P (1992) Chloroplast senescence. In NR Baker, HC Thomas, eds, Crop Photosynthesis. Elsevier, Amsterdam, pp413−440Google Scholar
  17. Meguro M, Ito H, Takabayashi A, Tanaka R, Tanaka A (2011) Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. Plant Cell 23:3442–3453CrossRefGoogle Scholar
  18. Morita R, Sato Y, Masuda Y, Nishimura M, Kusaba M (2009) Defect in non-yellow coloring 3, an α/β hydrolase-fold family protein, causes a stay-green phenotype during leaf senescence in rice. Plant J 59:940–952CrossRefGoogle Scholar
  19. Noodén LD (1988) The phenomena of senescence and aging. In LD Noodén, AC Leopold, eds, Senescence and aging in plants. Academic Press, London, pp1−50CrossRefGoogle Scholar
  20. Oberhuber M, Berghold J, Breuker K, Hörtensteiner S, Kraütler B (2003) Breakdown of chlorophyll:A nonenzymatic reaction accounts for the formation of the colorless “nonfluorescent” chlorophyll catabolites. Proc Natl Acad Sci USA 100:6910–6915CrossRefGoogle Scholar
  21. Oh MH, Kim YJ, Lee CH (2000) Leaf senescence in a stay-green mutant of Arabidopsis thaliana:disassembly process of photosystem I and II during dark-incubation. BMB Reports 33:256–262Google Scholar
  22. Oh MH, Moon YH, Lee CH (2003) Increased stability of LHCII by aggregate formation during dark-induced leaf senescence in the Arabidopsis mutant, ore10. Plant Cell Physiol 44:1368–1377CrossRefGoogle Scholar
  23. Oh MH, Kim JH, Moon YH, Lee CH (2004) Defects in a proteolytic step of light-harvesting complex II in an Arabidopsis stay-green mutant, ore10, during dark-induced leaf senescence. J Plant Biol 47:330–337CrossRefGoogle Scholar
  24. Oh MH, Safarova RB, Eu YJ, Zulfugarov IS, Kim JH, Hwang HJ, Lee CB, Lee CH (2009) Loss of peripheral polypeptides in the stromal side of photosystem I by light-chilling in cucumber leaves. Photochem Photobiol Sci 8:535–541CrossRefGoogle Scholar
  25. Park SY, Yu JW, Park JS, Li J, Yoo SC, Lee NY, Lee SK, Jeong SW, Seo HS, Koh HJ, Jeon JS, Park YI, Paek NC (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664CrossRefGoogle Scholar
  26. 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 Biophys Acta 975:384–394CrossRefGoogle Scholar
  27. Pružinská A, Tanner G, Anders I, Roca M, Hörtensteiner S (2003) Chlorophyll breakdown:pheophorbide a oxygenase is a Riesketype iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci USA 100:15259–15264CrossRefGoogle Scholar
  28. Pružinská A, Tanner G, Aubry S, Anders I, Moser S, Müller T, Ongania KH, Kräutler B, Youn JY, Liljegren SJ, Hörtensteiner S (2005) Chlorophyll breakdown in senescent Arabidopsis leaves. Characterization of chlorophyll catabolites and chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol 139:52–63Google Scholar
  29. Pružinská A, Anders I, Aubry S, Schenk N, Tapernoux-Lüthi E, Müller T, Kräutler B, Hörtensteiner S (2007) In vivo participation of red chlorophyll catabolite reductase in chlorophyll breakdown. Plant Cell 19:369–387CrossRefGoogle Scholar
  30. Ren G, Zhou Q, Wu S, Zhang Y, Zhang L, Huang J, Sun Z, Kuai B (2010) Reverse genetic identification of CRN1 and its distinctive role in chlorophyll degradation in Arabidopsis. JIPB 52:496–504Google Scholar
  31. Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76CrossRefGoogle Scholar
  32. Sakuraba Y, Schelbert S, Park SY, Han SH, Lee BD, Andrès CB, Kessler F, Hörtensteiner S, Paek NC (2012) STAY-GREEN and chlorophyll catabolic enzymes interact at light-harvesting complex II for chlorophyll detoxification during leaf senescence in Arabidopsis. Plant Cell 24:507–518CrossRefGoogle Scholar
  33. Sakuraba Y, Kim YS, Yoo SC, Hörtensteiner S, Paek NC (2013) 7-Hydroxymethyl chlorophyll a reductase functions in metabolic channeling of chlorophyll breakdown intermediates during leaf senescence. Biochem Biophys Res Commun 430:32–37Google Scholar
  34. Sato Y, Morita R, Katsuma S, Nishimura M, Tanaka A, Kusaba M (2009) Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice. Plant J 57:120–131CrossRefGoogle Scholar
  35. Schelbert S, Aubry S, Burla B, Agne B, Kessler F, Krupinska K, Hörtensteiner S (2009) Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. Plant Cell 21:767–785CrossRefGoogle Scholar
  36. Schenk N, Schelbert S, Kanwischer M, Goldschmidt EE, Dörmann P, Hörtensteiner S (2007) The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana. FEBS Lett 581:5517–5525CrossRefGoogle Scholar
  37. Scheumann V, Schoch S, Rüdiger W (1998) Chlorophyll a formation in the chlorophyll b reductase reaction requires reduced ferredoxin. J Biol Chem 273:35102–35108CrossRefGoogle Scholar
  38. Shimoda Y, Ito H, Tanaka A (2016) Arabidopsis STAY-GREEN, Mendel’s green cotyledon gene, encodes magnesium-dechelatase. Plant Cell 28:2147–2160CrossRefGoogle Scholar
  39. Smart CM (1994) Gene expression during leaf senescence. New Phytol 126:419–448CrossRefGoogle Scholar
  40. Suzuki T, Kunieda T, Murai F, Morioka S, Shioi Y (2005) Mgdechelation activity in radish cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a. Plant Physiol Biochem 43:459–464CrossRefGoogle Scholar
  41. Takamiya K, Tsuchiya T, Ohta H (2000) Degradation pathway(s) of chlorophyll:what has gene cloning revealed? Trends Plant Sci 5:426–431CrossRefGoogle Scholar
  42. Tanaka R, Hirashima M, Satoh S, Tanaka A (2003) The Arabidopsisaccelerated cell death gene ACD1 is involved in oxygenation of pheophorbide a:Inhibition of the pheophorbide a oxygenase activity does not lead to the “stay-green” phenotype in Arabidopsis. Plant Cell Physiol 44:1266–1274CrossRefGoogle Scholar
  43. Thimann KV (1980) The senescence of leaves. In KV Thimann, eds, Senescence in plants. CRC Press Inc., Boca Raton, pp 85−115Google Scholar
  44. Thomas H, Stoddart JL (1980) Leaf senescence. Annu Rev Plant Physiol 31:83–111CrossRefGoogle Scholar
  45. Tsuchiya T, Ohta H, Okawa K, Iwamatsu A, Shimada H, Masuda T, Takamiya K (1999) Cloning of chlorophyllase, the key enzyme in chlorophyll degradation:finding of a lipase motif and the induction by methyl jasmonate. Proc Natl Acad Sci USA 96:15362–15367CrossRefGoogle Scholar
  46. Yao N, Greenberg JT (2006) Arabidopsis ACCELERATED CELL DEATH2 modulates programmed cell death. Plant Cell 18:397–411CrossRefGoogle Scholar
  47. Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using floral dip method. Nat Protoc 1:641–646CrossRefGoogle Scholar

Copyright information

© Korean Society of Plant Biologists and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Young Nam Yang
    • 1
  • Rana B. Safarova
    • 1
  • So-Yon Park
    • 2
  • Yasuhito Sakuraba
    • 2
  • Min-Hyuk Oh
    • 1
    • 6
  • Ismayil S. Zulfugarov
    • 1
  • Chin Bum Lee
    • 3
  • Ayumi Tanaka
    • 4
  • Nam-Chon Paek
    • 2
    • 5
    Email author
  • Choon-Hwan Lee
    • 1
    Email author
  1. 1.Department of Integrated Biological Science and Department of Molecular BiologyPusan National UniversityBusanKorea
  2. 2.Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life SciencesSeoul National UniversitySeoulKorea
  3. 3.Department of Molecular BiologyDong-eui UniversityBusanKorea
  4. 4.Institute of Low Temperature ScienceHokkaido UniversitySapporoJapan
  5. 5.Crop Biotechnology Institute, Institutes of Green Bio Science and TechnologySeoul National UniversityPyeongchangKorea
  6. 6.Animal and Plant Quarantine AgencyYeongnam Regional OfficeBusanKorea

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