, Volume 241, Issue 6, pp 1497–1508 | Cite as

Generation of reactive oxygen species in thylakoids from senescing flag leaves of the barley varieties Lomerit and Carina

  • Anja Krieger-LiszkayEmail author
  • Mirl Trösch
  • Karin Krupinska
Original Article


Main conclusion

During senescence, production of reactive oxygen species increased in thylakoids. In two barley varieties, no difference in superoxide production was observed while singlet oxygen production increased only in one variety.


During senescence, chlorophyll content decreased and photosynthetic electron transport was inhibited as shown for flag leaves collected from barley varieties Lomerit and Carina grown in the field. Spin trapping electron paramagnetic resonance (EPR) was used to investigate the production of reactive oxygen species in thylakoid membranes during senescence. EPR measurements were performed with specific spin traps to discriminate between singlet oxygen on one hand and reactive oxygen intermediates on the other hand. The results show that the generation of reactive oxygen intermediates increases in both varieties during senescence. Singlet oxygen increased only in the variety cv. Lomerit while it remained constant at a low level in the variety cv. Carina. Measurements in the presence of inhibitors of photosystem II and of the cytochrome b6f complex revealed that in senescing leaves reduction of oxygen at the acceptor side of photosystem I was the major, but not the only source of superoxide anions. This study shows that during senescence the production of individual reactive oxygen species varies in different barley varieties.


Spin trapping electron paramagnetic resonance Reactive oxygen species Thylakoids Photosynthetic electron transport Barley 





3-(3,4-Dichlorophenyl)-1,1-dimethyl urea


2-Iodo-2′,4′,4′-trinitro-3-methyl-6-isopropyl diphenyl ether


Electron paramagnetic resonance


Light harvesting complex






Reactive oxygen species


Superoxide dismutase


2,2,6,6-Tetramethyl-4-piperidone hydrochloride



We thank Rüdiger Stroeh (farm manager of Hohenschulen, CAU, Kiel, Germany) and his co-workers for preparation of field plots. We also thank the early stage researchers Wera Kucharewicz and Aditi Das of the EU Marie Curie project “Croplife” (ITN: PITN-GA-2010-264394) for collecting samples and Luca Boschian for preparation of chloroplasts in 2013. DEPMPO was a kind gift of S. Pietri, Univeristé Aix-Marseille, France. This work was supported by the German Research Foundation (DFG) for financial support (KR1350/13-1, KR1350/14-1).

Supplementary material

425_2015_2274_MOESM1_ESM.pptx (248 kb)
Supplementary material 1 (PPTX 247 kb)


  1. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Ann Rev Plant Biol 55:373–399CrossRefGoogle Scholar
  2. Arato A, Bondarava N, Krieger-Liszkay A (2004) Production of reactive oxygen species in chloride- and calcium-depleted photosystem II and their involvement in photoinhibition. Biochim Biophys Acta Bioenerg 1608:171–180CrossRefGoogle Scholar
  3. Asada K, Kiso K, Yoshika WK (1974) Univalent reduction of molecular-oxygen by spinach-chloroplasts on illumination. J Biol Chem 249:2175–2181PubMedGoogle Scholar
  4. Baniulis D, Hasan SS, Stofleth JT, Cramer WA (2013) Mechanism of Enhanced superoxide production in the cytochrome b(6)f complex of oxygenic photosyn. Biochem 52:8975–8983CrossRefGoogle Scholar
  5. Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG, Lin JF, Wu SH, Swidzinski J, Ishizaki K, Leaver C (2005) Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J 42:567–585CrossRefPubMedGoogle Scholar
  6. Casano LM, Martin M, Sabater B (1994) Sensitivity of superoxide dismutase transcript levels and activities to oxidative stress is lower in mature-senescent than in young barley leaves. Plant Physiol 106:1033–1039PubMedCentralPubMedGoogle Scholar
  7. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ (2003) Production of reactive oxygen species by mitochondria—central role of complex III. J Biol Chem 278:36027–36031CrossRefPubMedGoogle Scholar
  8. Coste S, Baraloto C, Leroy C, Marcon E, Renaud A, Richardson AD, Roggy JC, Schimann H, Uddling J, Hérault B (2010) Assessing foliar chlorophyll contents with the SPAD-502 chlorophyll meter: a calibration test with thirteen tree species of tropical rainforest in French Guiana. Ann For Sci 67:607. doi: 10.1051/forest/2010020 CrossRefGoogle Scholar
  9. del Rio LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F, Corpas FJ, Jiménez A, López-Huertas E, Hernández JA (1998) The activated oxygen role of peroxisomes in senescence. Plant Physiol 116:1195–1200CrossRefPubMedCentralPubMedGoogle Scholar
  10. Dhindsa RS, Plumbdhindsa P, Thorpe TA (1981) Leaf senescence—correlated with increased levels of membrane-permeability and lipid-peroxidation, and decreased levels of superoxide-dismutase and catalase. J Exp Bot 32:93–101CrossRefGoogle Scholar
  11. Dhindsa RS, Plumbdhindsa PL, Reid DM (1982) Leaf senescence and lipid peroxidation—effects of some phytohormones, and scavengers of free-radicals and singlet oxygen. Physiol Plant 56:453–457CrossRefGoogle Scholar
  12. Edge R, Truscott T (2010) Properties of carotenoid radicals and excited states and their potential role in biological systems. In: Landrum J (ed) Carotenoids: physical, chemical, and biological functions and properties. CRC Press, Boca Raton, pp 283–307Google Scholar
  13. Finkelstein E, Rosen GM, Raukman EJ (1982) Production of hydroxyl radicals by decomposition of superoxide spin trapped adducts. Mol Pharmacol 21:262–265PubMedGoogle Scholar
  14. Fischer BB, Hideg E, Krieger-Liszkay A (2013) Production, detection, and signaling of singlet oxygen in photosynthetic organisms. Antiox Redox Signaling 18:2145–2162CrossRefGoogle Scholar
  15. Foyer CH, Noctor G (2013) Redox signaling in plants. Antiox Redox Signal 18:2087–2090CrossRefGoogle Scholar
  16. Frejaville C, Karoui H, Tuccio B, Lemoigne F, Culcasi M, Pietri S, Lausicella R, Tordo P (1995) 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide—a neW efficient phosphorylated Nitrone for the in vitro and in vivo spin-trapping of oxygen-centered radicals. J Med Chem 38:258–265CrossRefPubMedGoogle Scholar
  17. Gollmer A, Arnbjerg J, Blaikie FH, Pedersen BW, Breitenbach T, Daasbjerg K, Glasius M, Ogilby PR (2011) Singlet oxygen sensor green. Photochem Photobiol 87:671–6799. doi: 10.1111/j.1751-1097.2011.00900.x CrossRefPubMedGoogle Scholar
  18. Gregersen PL, Culetic A, Boschian L, Krupinska K (2013) Plant senescence and crop productivity. Plant Mol Biol 82:603–622CrossRefPubMedGoogle Scholar
  19. Heyno E, Gross CM, Laureau C, Culcasi M, Pietri S, Krieger-Liszkay A (2009) Plastid alternative oxidase (PTOX) promotes oxidative stress when overexpressed in tobacco. J Biol Chem 284(45):31174–31180. doi: 10.1074/jbc.M109.021667 CrossRefPubMedCentralPubMedGoogle Scholar
  20. Hideg E, Deák Z, Hakala-Yatkin M, Karonen M, Rutherford AW, Tyystjärvi E, Vass I, Krieger-Liszkay A (2011) Pure forms of the singlet oxygen sensors TEMP and TEMPD do not inhibit Photosystem II. Biochim Biophys Acta 1807:1658–1661CrossRefPubMedGoogle Scholar
  21. Hilditch P, Thomas H, Rogers LJ (1986) 2 Processes for the breakdown of the qb protein of chloroplasts. FEBS Lett 208:313–316CrossRefGoogle Scholar
  22. Hopkins M, Taylor C, Liu ZD, Ma FS, McNamara L, Wang TW, Thompson JE (2007) Regulation and execution of molecular disassembly and catabolism during senescence. New Phytol 175:201–214CrossRefPubMedGoogle Scholar
  23. Humbeck K, Quast S, Krupinska K (1996) Functional and molecular changes in the photosynthetic apparatus during senescence of flag leaves from field-grown barley plants. Plant Cell Environ 19:337–344CrossRefGoogle Scholar
  24. Juvany M, Müller M, Munné-Bosch S (2013) Photo-oxidative stress in emerging and senescing leaves: a mirror image? J Exp Bot 64:3087–3098CrossRefPubMedGoogle Scholar
  25. Kar M, Feierabend J (1984) Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence. Planta 160:385–391CrossRefPubMedGoogle Scholar
  26. Karpinski S, Szechynska-Hebda M, Wituszynska W, Burdiak P (2013) Light acclimation, retrograde signalling, cell death and immune defences in plants. Plant Cell Environ 36:736–744CrossRefPubMedGoogle Scholar
  27. Kim C, Apel K (2013) Singlet oxygen-mediated signaling in plants: moving from flu to wild type reveals an increasing complexity. Photosyn Res 116:455–464CrossRefPubMedGoogle Scholar
  28. Kim C, Meskauskiene R, Zhang S, Lee KP, Lakshmanan Ashok M, Blajecka K, Herrfurth C, Feussner I, Apel K (2012) Chloroplasts of Arabidopsis are the source and a primary target of a plant-specific programmed cell death signaling pathway. Plant Cell 7:3026–3039CrossRefGoogle Scholar
  29. Krupinska K, Humbeck K (2004) Photosynthesis and chloroplast breakdown. In: Noóden LD (ed) Plant cell death processes. Elsevier Academic Press, San Diego, pp 169–188CrossRefGoogle Scholar
  30. Krupinska K, Mulisch M, Hollmann J, Tokarz K, Zschiesche W, Kage H, Humbeck K, Bilger W (2012) An alternative strategy of dismantling of the chloroplasts during leaf senescence observed in a high-yield variety of barley. Physiol Plant 144:189–200CrossRefPubMedGoogle Scholar
  31. Kurepa J, Herouart D, VanMontagu M, Inze D (1997) Differential expression of CuZn- and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments. Plant Cell Physiol 38:463–470CrossRefPubMedGoogle Scholar
  32. Li ZR, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and Responding to Excess Light. Ann Rev Plant Biol 60:239–260CrossRefGoogle Scholar
  33. McRae DG, Thompson JE (1983) Senescence-dependent changes in superoxide anion production by illuminated chloroplasts from bean-leaves. Planta 158:185–193CrossRefPubMedGoogle Scholar
  34. Mehta RA, Fawcett TW, Porath D, Mattoo AK (1992) Oxidative stress causes rapid membrane translocation and in vivo degradation of Ribulose-1,5-bisphosphate carboxylase oxygenase. J Biol Chem 267:2810–2816PubMedGoogle Scholar
  35. Miersch I, Heise J, Zelmer I, Humbeck K (2000) Differential degradation of the photosynthetic apparatus during leaf senescence in barley (Hordeum vulgare L.). Plant Biol 2:618–623CrossRefGoogle Scholar
  36. Mubarakshina MM, Ivanov BN (2010) The production and scavenging of reactive oxygen species in the plastoquinone pool of chloroplast thylakoid membranes. Physiol Plant 140:103–110CrossRefPubMedGoogle Scholar
  37. Mulisch M, Krupinska K (2013) Ultrastructural analyses of senescence associated dismantling of chloroplasts revisited. In: Biswal B, Krupinska K, Biswal UC (eds) Plastid development in leaves during growth and senescence. Springer, Dordrecht, pp 529–550Google Scholar
  38. Mullineaux PM, Baker NR (2010) Oxidative stress: antagonistic signaling for acclimation or cell death? Plant Physiol 154:521–525CrossRefPubMedCentralPubMedGoogle Scholar
  39. Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1–13CrossRefPubMedCentralPubMedGoogle Scholar
  40. Navabpour S, Morris K, Allen R, Harrison E, A-H-Mackerness S, Buchanan-Wollaston V (2003) Expression of senescence-enhanced genes in response to oxidative stress. J Exp Bot 54:2285–2292CrossRefPubMedGoogle Scholar
  41. Ohe M, Rapolu M, Mieda T, Miyagawa Y, Yabuta Y, Yoshimura K, Shigeoka S (2005) Decline in leaf photooxidative-stress tolerance with age in tobacco. Plant Sci 168:1487–1493CrossRefGoogle Scholar
  42. Pastori GM, del Rio LA (1997) Natural senescence of pea leaves—an activated oxygen-mediated function for peroxisomes. Plant Physiol 113:411–418PubMedCentralPubMedGoogle Scholar
  43. Pintó-Marijuan M, Munné-Bosch S (2014) Photo-oxidative stress markers as a measure of abiotic stress-induced leaf senescence/advantages and limitations. J Exp Bot 65:3845–3857CrossRefPubMedGoogle Scholar
  44. 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
  45. Pospisil P (2012) Molecular mechanisms of production and scavenging of reactive oxygen species by photosystem II. Biochim Biophys Acta 1817:218–231CrossRefPubMedGoogle Scholar
  46. Pospisil P, Arato A, Krieger-Liszkay A, Rutherford AW (2004) Hydroxyl radical generation by photosystem II. Biochem 43:6783–6792CrossRefGoogle Scholar
  47. Prochazkova D, Wilhelmova N (2007) Leaf senescence and activities of the antioxidant enzymes. Biol Plant 51:401–406CrossRefGoogle Scholar
  48. Prochazkova D, Sairam RK, Srivastava GC, Singh DV (2001) Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Sci 161:765–771CrossRefGoogle Scholar
  49. Ramos CL, Pou S, Britigan BE, Cohen MS, Rosen GM (1992) Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes. J Biol Chem 267:8307–8312PubMedGoogle Scholar
  50. Rinalducci S, Pedersen JZ, Zolla L (2004) Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II. Biochim Biophys Acta 1608:63–73CrossRefPubMedGoogle Scholar
  51. Rosenwasser S, Rot I, Sollner E, Meyer AJ, Smith Y, Leviatan N, Fluhr R, Friedman H (2011) Organelles contribute differentially to reactive oxygen species-related events during extended darkness. Plant Physiol 156:185–201CrossRefPubMedCentralPubMedGoogle Scholar
  52. Rutherford AW, Krieger-Liszkay A (2001) Herbicide-induced oxidative stress in photosystem II. Trends Biochem Sci 26:648–653CrossRefPubMedGoogle Scholar
  53. Sabater B, Martín M (2013) Chloroplast control of leaf senescence. In: Biswal B, Krupinska K, Biswal UC (eds) Plastid development in leaves during growth and senescence. Springer, Dordrecht, pp 529–550CrossRefGoogle Scholar
  54. Schröder WP, Akerlund HE (1986) H2O2 accessibility to the photosystem-II donor side in protein-depleted inside-out thylakoids measured as flash-induced oxygen production. Biochim Biophys Acta 848:359–363CrossRefGoogle Scholar
  55. Tang YL, Wen XG, Lu CM (2005) Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice. Plant Physiol Biochem 43:193–201CrossRefPubMedGoogle Scholar
  56. Vanacker H, Sandalio LM, Jimenez A, Palma JM, Corpas FJ, Meseguer V, Gomez M, Sevilla F, Leterrier M, Foyer CH, del Rio LA (2006) Roles for redox regulation in leaf senescence of pea plants grown on different sources of nitrogen nutrition. J Exp Bot 57:1735–1745CrossRefPubMedGoogle Scholar
  57. Yu Q, Feilke K, Krieger-Liszkay A, Beyer P (2014) Functional and molecular characterization of plastid terminal oxidase from rice (Oryza sativa). Biochim Biophys Acta 1837:1284–1292CrossRefPubMedGoogle Scholar
  58. Zapata JM, Guéra A, Esteban-Carrasco A, Martín M, Sabater B (2005) Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco. Cell Death Differ 12:1277–1284CrossRefPubMedGoogle Scholar
  59. Zimmermann P, Zentgraf U (2005) The correlation between oxidative stress and leaf senescence during plant development. Cell Mol Biol Lett 10:515–534PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Anja Krieger-Liszkay
    • 1
    Email author
  • Mirl Trösch
    • 2
  • Karin Krupinska
    • 2
  1. 1.Institute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA) Saclay, Institut de Biologie et de Technologie de Saclay, Centre National de la Recherche Scientifique (CNRS)Université Paris-SudGif-sur-Yvette cedexFrance
  2. 2.Institute of BotanyUniversity of KielKielGermany

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