Plant Molecular Biology

, Volume 67, Issue 3, pp 243–256 | Cite as

Stay-green protein, defective in Mendel’s green cotyledon mutant, acts independent and upstream of pheophorbide a oxygenase in the chlorophyll catabolic pathway

  • Sylvain Aubry
  • Jan Mani
  • Stefan HörtensteinerEmail author


Type C stay-green mutants are defined as being defective in the pathway of chlorophyll breakdown, which involves pheophorbide a oxygenase (PAO), required for loss of green color. By analyzing senescence parameters, such as protein degradation, expression of senescence-associated genes and loss of photosynthetic capacity, we demonstrate that JI2775, the green cotyledon (i) pea line used by Gregor Mendel to establish the law of genetics, is a true type C stay-green mutant. STAY-GREEN (SGR) had earlier been shown to map to the I locus. The defect in JI2775 is due to both reduced expression of SGR and loss of SGR protein function. Regulation of PAO through SGR had been proposed. By determining PAO protein abundance and activity, we show that PAO is unaffected in JI2775. Furthermore we show that pheophorbide a accumulation in the mutant is independent of PAO. When silencing SGR expression in Arabidopsis pao1 mutant, both pheophorbide a accumulation and cell death phenotype, typical features of pao1, are lost. These results confirm that SGR function within the chlorophyll catabolic pathway is independent and upstream of PAO.


Chlorophyll breakdown Gregor Mendel Pea I locus Pheophorbide a oxygenase Stay-green mutant Senescence 







Expressed sequence tags


Light harvesting complex of photosystem II


Nonfluorescent chlorophyll catabolites




Pheophorbide a oxygenase


Primary fluorescent chlorophyll catabolite




Red chlorophyll catabolite reductase





We thank B. Burla, University of Zurich, for his help with phylogenetic analyses. We thank S. Schelbert, University of Zurich, for critical reading of the manuscript and I. Anders, University of Bern, for technical support. This work was supported by the Swiss National Science Foundation (3100A0-105389) and the National Center of Competence in Research Plant Survival, research program of the Swiss National Science Foundation.

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  1. Akhtar MS, Goldschmidt EE, John I, Rodoni S, Matile P, Grierson D (1999) Altered patterns of senescence and ripening in gf, a stay-green mutant of tomato (Lycopersicon esculentum Mill.). J Exp Bot 50:1115–1122CrossRefGoogle Scholar
  2. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmermann J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Chory N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657PubMedCrossRefGoogle Scholar
  3. Armstead I, Donnison I, Aubry S, Harper J, Hörtensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2006) From crop to model to crop: identifying the genetic basis of the staygreen mutation in the Lolium/Festuca forage and amenity grasses. New Phytol 172:592–597PubMedCrossRefGoogle Scholar
  4. Armstead I, Donnison I, Aubry S, Harper J, Hörtensteiner S, James C, Mani J, Moffet M, Ougham H, Roberts L, Thomas A, Weeden N, Thomas H, King I (2007) Cross-species identification of Mendel’s I locus. Science 315:73PubMedCrossRefGoogle Scholar
  5. Bachmann A, Fernández-López J, Ginsburg S, Thomas H, Bouwcamp JC, Solomos T, Matile P (1994) Stay-green genotypes of Phaseolus vulgaris L.: chloroplast proteins and chlorophyll catabolites during foliar senescence. New Phytol 126:593–600CrossRefGoogle Scholar
  6. Bhattacharyya MK, Smith AM, Ellis THN, Hedley C, Martin C (1990) The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell 60:115–122PubMedCrossRefGoogle Scholar
  7. Chakraborti D, Sarkar A, Gupta S, Das S (2006) Small and large scale genomic DNA isolation protocol for chickpea (Cicer arietinum L.), suitable for molecular marker and transgenic analyses. Afr J Biotechnol 5:585–589Google Scholar
  8. Chung DW, Pružinská A, Hörtensteiner S, Ort DR (2006) The role of pheophorbide a oxygenase expression and activity in the canola green seed problem. Plant Physiol 142:88–97PubMedCrossRefGoogle Scholar
  9. Costa ML, Civello PM, Chaves AR, Martinez GA (2002) Characterization of Mg-dechelatase activity obtained from Fragaria × ananassa fruit. Plant Physiol Biochem 40:111–118CrossRefGoogle Scholar
  10. Efrati A, Eyal Y, Paran I (2005) Molecular mapping of the chlorophyll retainer (cl) mutation in pepper (Capsicum spp.) and screening for candidate genes using tomato ESTs homologous to structural genes of the chlorophyll catabolism pathway. Genome 48:347–351PubMedGoogle Scholar
  11. Gray J, Wardzala E, Yang M, Reinbothe S, Haller S, Pauli F (2004) A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers. Plant Mol Biol 54:39–54PubMedCrossRefGoogle Scholar
  12. Havaux M (1993) Characterization of thermal damage to the photosynthetic electron transport system in potato leaves. Plant Sci 94:19–33CrossRefGoogle Scholar
  13. Hellens R, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832PubMedCrossRefGoogle Scholar
  14. Hilditch P, Thomas H, Rogers L (1986) Leaf senescence in a non-yellowing mutant of Festuca pratensis: Photosynthesis and photosynthetic electron transport. Planta 167:146–151CrossRefGoogle Scholar
  15. Hilditch PI, Thomas H, Thomas BJ, Rogers LJ (1989) Leaf senescence in a non-yellowing mutant of Festuca pratensis: proteins of photosystem II. Planta 177:265–272CrossRefGoogle Scholar
  16. Horn R, Paulsen H (2004) Early steps in the assembly of light-harvesting chlorophyll a/b complex - Time-resolved fluorescence measurements. J Biol Chem 279:44400–44406PubMedCrossRefGoogle Scholar
  17. Hörtensteiner S (2006) Chlorophyll degradation during senescence. Annu Rev Plant Biol 57:55–77PubMedCrossRefGoogle Scholar
  18. Hörtensteiner S, Vicentini F, 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
  19. Hörtensteiner S, Chinner J, Matile P, Thomas H, Donnison IS (2000) Chlorophyll breakdown in Chlorella protothecoides: characterization of degreening and cloning of degreening-related genes. Plant Mol Biol 42:439–450PubMedCrossRefGoogle Scholar
  20. Jiang H, Li M, Liang N, Yan H, Wei Y, Xu X, Liu J, Xu Z, Chen F, Wu G (2007) Molecular cloning and function analysis of the stay green gene in rice. Plant J 52:197–209PubMedCrossRefGoogle Scholar
  21. Kingston-Smith AH, Thomas H, Foyer CH (1997) Chlorophyll a fluorescence, enzyme and antioxidant analyses provide evidence for the operation of alternative electron sinks during leaf senescence in a stay-green mutant of Festuca pratensis. Plant Cell Environ 20:1323–1337CrossRefGoogle Scholar
  22. Kreuz K, Tommasini R, Martinoia E (1996) Old enzymes for a new job. Herbicide detoxification in plants. Plant Physiol 111:349–353PubMedGoogle Scholar
  23. Kürsteiner O, Dupuis I, Kuhlemeier C (2003) The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses. Plant Physiol 132:968–978PubMedCrossRefGoogle Scholar
  24. 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 19:1362–1375PubMedCrossRefGoogle Scholar
  25. Lester DR, Ross JJ, Davies PJ, Reid JB (1997) Mendel’s stem length gene (Le) encodes a gibberellin 3β-hydroxylase. Plant Cell 9:1435–1443PubMedCrossRefGoogle Scholar
  26. Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA 97:13401–13406PubMedCrossRefGoogle Scholar
  27. Matile P, Hörtensteiner S, Thomas H (1999) Chlorophyll degradation. Annu Rev Plant Physiol Plant Mol Biol 50:67–95PubMedCrossRefGoogle Scholar
  28. Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51:659–668PubMedCrossRefGoogle Scholar
  29. Mendel G (1866) Versuche über Pflanzenhybriden. Verh Naturforsch Ver Brünn 4:3–47Google Scholar
  30. Müller T, Moser S, Ongania K-H, Pružinská A, Hörtensteiner S, Kräutler B (2006) A divergent path of chlorophyll breakdown in the model plant Arabidopsis thaliana. ChemBioChem 7:40–42PubMedCrossRefGoogle Scholar
  31. Murray MG, Thompson WF (1980) Rapid isolation of high molecular-weight plant DNA. Nucl Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  32. Nott A, Jung HS, Koussevitzky S, Chory J (2006) Plastid-to-nucleus retrograde signaling. Annu Rev Plant Biol 57:739–759PubMedCrossRefGoogle Scholar
  33. Oberhuber M, Berghold J, Breuker K, Hörtensteiner S, Kräutler B (2003) Breakdown of chlorophyll: a nonenzymatic reaction accounts for the formation of the colorless “nonfluorescent” chlorophyll catabolites. Proc Natl Acad Sci USA 100:6910–6915PubMedCrossRefGoogle Scholar
  34. 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. J Biochem Mol Biol 33:256–262Google Scholar
  35. Park S-Y, Yu J-W, Park J-S, Li J, Yoo S-C, Lee N-Y, Lee S-K, Jeong S-W, Seo HS, Koh H-J, Jeon J-S, Park Y-I, Paek N-C (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664PubMedCrossRefGoogle Scholar
  36. Pružinská A, Anders I, Tanner G, Roca M, Hörtensteiner S (2003) Chlorophyll breakdown: pheophorbide a oxygenase is a Rieske-type iron-sulfur protein, encoded by the accelerated cell death 1 gene. Proc Natl Acad Sci USA 100:15259–15264PubMedCrossRefGoogle Scholar
  37. Pružinská A, Tanner G, Aubry S, Anders I, Moser S, Müller T, Ongania K-H, Kräutler B, Youn J-Y, Liljegren SJ, Hörtensteiner S (2005) Chlorophyll breakdown in senescent Arabidopsis leaves: characterization of chlorophyll catabolites and of chlorophyll catabolic enzymes involved in the degreening reaction. Plant Physiol 139:52–63PubMedCrossRefGoogle Scholar
  38. 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 19:369–387PubMedCrossRefGoogle Scholar
  39. Quirino BF, Noh YS, Himelblau E, Amasino RM (2000) Molecular aspects of leaf senescence. Trends Plant Sci 5:278–282PubMedCrossRefGoogle Scholar
  40. Ren G, An K, Liao Y, Zhou X, Cao Y, Zhao H, Ge X, Kuai B (2007) Identification of a novel chloroplast protein AtNYE1 regulating chlorophyll degradation during leaf senescence in Arabidopsis. Plant Physiol 144:1429–1441PubMedCrossRefGoogle Scholar
  41. Roca M, James J, Pružinská A, Hörtensteiner S, Thomas H, Ougham H (2004) Analysis of the chlorophyll catabolism pathway in leaves of an introgression senescence mutant of Lolium temulentum. Phytochemistry 65:1231–1238PubMedCrossRefGoogle Scholar
  42. Sato Y, Morita R, Nishimura M, Yamaguchi H, Kusaba M (2007) Mendel’s green cotyledon gene encodes a positive regulator of the chlorophyll-degrading pathway. Proc Natl Acad Sci USA 104:14169–14174PubMedCrossRefGoogle Scholar
  43. Scheumann V, Schoch S, Rüdiger W (1999) Chlorophyll b reduction during senescence of barley seedlings. Planta 209:364–370PubMedCrossRefGoogle Scholar
  44. Sidler M, Hassa P, Hasan S, Ringli C, Dudler R (1998) Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light. Plant Cell 10:1623–1636PubMedCrossRefGoogle Scholar
  45. Strain HH, Cope BT, Svec WA (1971) Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. Methods Enzymol 23:452–476CrossRefGoogle Scholar
  46. Suzuki T, Kunieda T, Murai F, Morioka S, Shioi Y (2005) Mg-dechelation activity in radish cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a. Plant Physiol Biochem 43:459–464PubMedCrossRefGoogle Scholar
  47. Thomas H (1987) Sid: a Mendelian locus controlling thylakoid membrane disassembly in senescing leaves of Festuca pratensis. Theor Appl Genet 73:551–555CrossRefGoogle Scholar
  48. Thomas H (1997) Chlorophyll: a symptom and a regulator of plastid development. New Phytol 136:163–181CrossRefGoogle Scholar
  49. Thomas H, Howarth CJ (2000) Five ways to stay green. J Exp Bot 51:329–337PubMedCrossRefGoogle Scholar
  50. Thomas H, Schellenberg M, Vicentini F, Matile P (1996) Gregor Mendel’s green and yellow pea seeds. Bot Acta 109:3–4Google Scholar
  51. Vicentini F, Hörtensteiner S, Schellenberg M, Thomas H, Matile P (1995) Chlorophyll breakdown in senescent leaves: identification of the biochemical lesion in a stay-green genotype of Festuca pratensis Huds. New Phytol 129:247–252CrossRefGoogle Scholar
  52. Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbot D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throuput gene silencing in plants. Plant J 27:581–590PubMedCrossRefGoogle Scholar
  53. White OE (1916) Inheritance studies in Pisum I Inheritance of cotyledon color. Am Nat 50:530–547CrossRefGoogle Scholar
  54. Zelisko A, Garcia-Lorenzo M, Jackowski G, Jansson S, Funk C (2005) AtFtsH6 is involved in the degradation of the light-harvesting complex II during high-light acclimation and senescence. Proc Natl Acad Sci USA 102:13699–13704PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Sylvain Aubry
    • 1
  • Jan Mani
    • 2
    • 3
  • Stefan Hörtensteiner
    • 1
    • 2
    Email author
  1. 1.Institute of Plant BiologyUniversity of ZurichZurichSwitzerland
  2. 2.Institute of Plant SciencesUniversity of BernBernSwitzerland
  3. 3.Institute of Cell BiologyUniversity of BernBernSwitzerland

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