Acta Physiologiae Plantarum

, Volume 34, Issue 2, pp 617–629

Antioxidant system in programmed cell death of sycamore (Acer pseudoplatanus L.) cultured cells

  • Nicla Contran
  • Mariagrazia Tonelli
  • Paolo Crosti
  • Raffaella Cerana
  • Massimo Malerba
Original Paper


Reactive oxygen species (ROS) have pleiotropic effects in plants. ROS can lead to cellular damage and death or play key roles in control and regulation of biological processes, such as programmed cell death (PCD). This dual role of ROS, as toxic or signalling molecules, is possible because plant antioxidant system (AS) is able to achieve a tight control over ROS cellular levels, balancing properly their production and scavenging. AS response in plant PCD has been clearly described only in the hypersensitive response in incompatible plant–pathogen interactions and in the senescence process and has not been completely unravelled. In sycamore (Acer pseudoplatanus L.) cultured cells PCD can be induced by Fusicoccin (Fc), Tunicamycin (Tu), and Brefeldin A (Ba). These chemicals induce comparable PCD time course and extent, while H2O2 production is detectable only in Fc- and, to a lesser extent, in Ba-treated cells. In this paper the AS has been investigated during PCD of sycamore cells, measuring the effects of the three inducers on the cellular levels of non-enzymatic and enzymatic antioxidants. Results show that the AS behaviour is different in the PCD induced by the three chemicals. In Fc-treated cells AS is mainly devoted to decrease the concentration of toxic intracellular H2O2 levels. On the contrary, in cells treated with Tu and Ba, the cell redox state is shifted to a more reduced state and the enzymatic AS is partially down-regulated, allowing ROS to act as signalling molecules.


Reactive oxygen species Programmed cell death Antioxidant enzyme Ascorbate–glutathione cycle Acer pseudoplatanus L. 



Ascorbate peroxidase


Antioxidant system


Ascorbic acid


Brefeldin A




Dehydroascorbic acid


Dehydroascorbate reductase




Glutathione peroxidase


Glutathione reductase


Reduced glutathione


Oxidized glutathione


Hydrogen peroxide




Monodehydroascorbate reductase


Programmed cell death


Reactive oxygen species


Superoxide dismutase




  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126PubMedCrossRefGoogle Scholar
  2. Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedCrossRefGoogle Scholar
  3. Barth C, Moeder W, Klessig DF, Conklin PL (2004) The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1. Plant Physiol 134:1784–1792PubMedCrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Cacas JL (2010) Devil inside: does plant programmed cell death involve the endomembrane system? Plant Cell Environ 33:1453–1473PubMedGoogle Scholar
  6. Cakmak I, Strbac D, Marschner H (1993) Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds. J Exp Bot 44:127–132CrossRefGoogle Scholar
  7. Contran N, Cerana R, Crosti P, Malerba M (2007) Cyclosporin A inhibits programmed cell death and cytochrome c release induced by fusicoccin in sycamore cells. Protoplasma 231:193–199PubMedCrossRefGoogle Scholar
  8. Crosti P, Malerba M, Bianchetti R (2001) Tunicamycin and Brefeldin A induce in plant cells a programmed cell death showing apoptotic features. Protoplasma 216:31–38PubMedCrossRefGoogle Scholar
  9. Dangl JL, Dietrich RA, Thomas H (2000) Senescence and programmed cell death. In: Buchanan B, Gruissem W, Jones R (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists Press, Rockville, pp 1044–1100Google Scholar
  10. Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal interaction between nitric oxide and oxygen intermediates in the plant hypersensitive disease resistance response. Proc Natl Acad Sci USA 88:13454–13459CrossRefGoogle Scholar
  11. Elbein AD (1987) Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem 56:497–534PubMedCrossRefGoogle Scholar
  12. Florence TM (1980) Degradation of protein disulphide bonds in dilute alkali. Biochem J 189:507–520PubMedGoogle Scholar
  13. Foyer CH, Noctor G (2000) Oxygen processing in photosynthesis: regulation and signalling. New Phytol 146:359–388CrossRefGoogle Scholar
  14. Foyer CH, Noctor G (2005) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056–1071CrossRefGoogle Scholar
  15. Foyer CH, Noctor G (2009) Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications. Antioxid Redox Sign 11:861–905CrossRefGoogle Scholar
  16. Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol 155:2–18PubMedCrossRefGoogle Scholar
  17. Gechev TS, Van Breusegem F, Stone JM, Denev I, Laloi C (2006) Reactive oxygen species as signals that modulate plant stress responses and programmed cell death. Bioessays 28:1091–1101PubMedCrossRefGoogle Scholar
  18. Giannopolitis CN, Ries SK (1977) Superoxide dismutase I. Occurrence in higher plants. Plant Physiol 59:309–314PubMedCrossRefGoogle Scholar
  19. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930PubMedCrossRefGoogle Scholar
  20. Halliwell B (2003) Oxidative stress in cell culture: an under-appreciated problem? FEBS Lett 540:3–6PubMedCrossRefGoogle Scholar
  21. Halliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35:1147–1150PubMedCrossRefGoogle Scholar
  22. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:180–198Google Scholar
  23. Hissin PJ, Hilf R (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 74:214–226PubMedCrossRefGoogle Scholar
  24. Jacobson MD, Weil M, Raff MC (1997) Programmed cell death in animal development. Cell 88:347–354PubMedCrossRefGoogle Scholar
  25. Jiang M, Zhang J (2001) Effect of abscisic acid on active oxygen species, antioxidative defence system and oxidative damage in leaves of maize seedlings. Plant Cell Physiol 42:1265–1273PubMedCrossRefGoogle Scholar
  26. Kampfenkel K, Van Montagu M, Inzè D (1995) Extraction and determination of ascorbate and dehydroascorbate from plant tissue. Anal Biochem 225:165–167PubMedCrossRefGoogle Scholar
  27. Kanematsu S, Asada K (1990) Characteristic amino-acid-sequences of chloroplast and cytosol isozymes of CuZn-superoxide dismutase in spinach, rice and horsetail. Plant Cell Physiol 31:99–112Google Scholar
  28. Koshiba T (1993) Cytosolic ascorbate peroxidase in seedlings and leaves of maize (Zea mays). Plant Cell Physiol 34:713–721Google Scholar
  29. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  30. Lam E (2008) Programmed cell death in plants: orchestrating an intrinsic suicide program within walls. Crit Rev Plant Sci 27:413–423CrossRefGoogle Scholar
  31. Lawrence RA, Burk RF (1976) Glutathione peroxidase activity in selenium deficient rat liver. Biochem Biophys Res Commun 71:952–958PubMedCrossRefGoogle Scholar
  32. Li Z, Xing D (2011) Mechanistic study of mitochondria-dependent programmed cell death induced by aluminum phytotoxicity using fluorescence techniques. J Exp Bot 62:331–343PubMedCrossRefGoogle Scholar
  33. Mahalingam R, Fedoroff N (2003) Stress response, cell death and signalling: the many faces of reactive oxygen species. Physiol Plant 119:56–68CrossRefGoogle Scholar
  34. Malerba M, Cerana R, Crosti P, Bianchetti R (2003a) Fusicoccin stimulates the production of H2O2 in sycamore cell cultures and induces alternative respiration and cytochrome c leakage from mitochondria. Physiol Plant 119:480–488CrossRefGoogle Scholar
  35. Malerba M, Cerana R, Crosti P (2003b) Fusicoccin induces in plant cells a programmed cell death showing apoptotic features. Protoplasma 222:113–116PubMedCrossRefGoogle Scholar
  36. Malerba M, Cerana R, Crosti P (2004a) Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells. Protoplasma 224:61–70PubMedGoogle Scholar
  37. Malerba M, Crosti P, Cerana R, Bianchetti R (2004b) Fusicoccin affects cytochrome c leakage and cytosolic 14-3-3 accumulation independent of H+-ATPase activation. Physiol Plant 120:386–394PubMedCrossRefGoogle Scholar
  38. Malerba M, Crosti P, Cerana R (2005) The fusicoccin-induced accumulation of nitric oxide in sycamore cultured cells is not required for the toxin-stimulated stress-related responses. Plant Sci 168:381–387CrossRefGoogle Scholar
  39. Malerba M, Contran N, Tonelli M, Crosti P, Cerana R (2008) Role of nitric oxide in actin depolymerization and programmed cell death induced by fusicoccin in sycamore (Acer pseudoplatanus L.) cultured cells. Physiol Plant 133:449–457PubMedCrossRefGoogle Scholar
  40. Marrè E (1979) Fusicoccin: a tool in plant physiology. Annu Rev Plant Physiol 30:273–288CrossRefGoogle Scholar
  41. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410PubMedCrossRefGoogle Scholar
  42. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498PubMedCrossRefGoogle Scholar
  43. Morera C, Villanueva MA (2009) Heat treatment and viability assessment by Evans blue in cultured Symbiodinium kawagutii cells. World J Microbiol Biotechnol 25:1125–1128CrossRefGoogle Scholar
  44. Mou Z, Fan WH, Dong XN (2003) Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 113:935–944PubMedCrossRefGoogle Scholar
  45. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  46. Noctor G, Foyer C (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279CrossRefGoogle Scholar
  47. Orendi G, Zimmermann P, Baar C, Zentgraf U (2001) Loss of stress-induced expression of catalase3 during leaf senescence in Arabidopsis thaliana is restricted to oxidative stress. Plant Sci 161:301–314PubMedCrossRefGoogle Scholar
  48. Shulaev V, Oliver DJ (2006) Metabolic and proteomic markers for oxidative stress. New tools for reactive oxygen species research. Plant Physiol 141:367–372PubMedCrossRefGoogle Scholar
  49. Simons SSJ, Johnson DF (1978) Reaction of o-phthalaldehyde and thiols with primary amines: fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles. Anal Biochem 90:705–725PubMedCrossRefGoogle Scholar
  50. Staehelin LA, Driouich A (1997) Brefeldin A effects in plants. Plant Physiol 114:401–403PubMedGoogle Scholar
  51. Stein JC, Hansen G (1999) Mannose induces an endonuclease responsible for DNA laddering in plant cells. Plant Physiol 121:71–79PubMedCrossRefGoogle Scholar
  52. Ushimaru T, Ogawa K, Ishida N, Shibasaka M, Kanematsu S, Asada K, Tsuji H (1995) Changes in organelle superoxide-dismutase isoenzymes during air adaptation of submerged rice seedlings—differential behaviour of isoenzymes in plastids and mitochondria. Planta 196:606–613CrossRefGoogle Scholar
  53. Van Breusegem F, Dat JF (2006) Reactive oxygen species in plant cell death. Plant Physiol 141:384–390PubMedCrossRefGoogle Scholar
  54. Wendehenne D, Durner J, Klessig DF (2004) Nitric oxide: a new player in plant signalling and defence responses. Curr Opin Plant Biol 7:449–455PubMedCrossRefGoogle Scholar
  55. Ye ZZ, Rodriguez R, Tran A, Hoang H, de los Santos D, Brown S, Vellanoweth RL (2000) The developmental transition to flowering represses ascorbate peroxidase activity and induces enzymatic lipid peroxidation in leaf tissue in Arabidopsis thaliana. Plant Sci 158:115–127PubMedCrossRefGoogle Scholar
  56. Zhang JX, Kirkham MB (1996) Antioxidant responses to drought in sunflower and sorghum seedlings. New Phytol 132:361–373CrossRefGoogle Scholar
  57. Zhang LR, Xing D (2008) Methyl jasmonate induces production of reactive oxygen species and alterations in mitochondrial dynamics that precede photosynthetic dysfunction and subsequent cell death. Plant Cell Physiol 49:1092–1111PubMedCrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2011

Authors and Affiliations

  • Nicla Contran
    • 1
  • Mariagrazia Tonelli
    • 1
  • Paolo Crosti
    • 1
  • Raffaella Cerana
    • 2
  • Massimo Malerba
    • 1
  1. 1.Department of Biotechnology and BiosciencesUniversity of Milano-BicoccaMilanItaly
  2. 2.Department of Environmental SciencesUniversity of Milano-BicoccaMilanItaly

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