Abstract
In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H2O2 and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H2O2 was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.
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Abbreviations
- APX:
-
ascorbate peroxidase
- ASC:
-
ascorbic acid
- CAT:
-
catalase
- FW:
-
fresh weight
- GPX:
-
guaiacol peroxidase
- ROS:
-
reactive oxygen species
- SOD:
-
superoxide dismutase
References
Aebi H. 1984. Catalase in vitro. In: Colowick, S.P., Kaplan, N.O., (Eds), Methods Enzymol., Acad. Press. Florida, 105: 121–126.
Alexieva V., Sergiev I, Mapelli S., Karanov E. 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell Environ. 24: 1337–1344.
Alscher R.G., Erturk N., Heath L.S. 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot., 53: 1331–1341.
Arnon D. I. 1949. Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris. Plant Physiol., 24: 1–15.
Biswal B., Biswal U. C. 1999. Leaf senescence: Physiology and molecular biology. Cur. Sci., 77: 755–782.
Bradford M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248–254.
Beauchamp Ch., Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276–287.
Chang C. J., Kao C. H. 1998. H2O2 metabolism during senescence of rice leaves: changes in enzyme activities in light and darkness. Plant Growth Regul., 25: 11–15.
Chiba A., Ishida H., Nishizawa N. K., Makino A., Mae T. 2003. Exclusion of ribulose-1,5-bisphosphate carboxylase/oxygenase from chloroplasts by specific bodies in naturally senescing leaves of wheat. Plant Cell Physiol. 44: 914–921.
Dat J., Vandanabeele S., Vranova E., Van Montagu M., Inze D., Van Breusegem F. 2000. Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sc., 57: 779–795.
De Kok L.J., Graham M. 1989. Levels of pigments, soluble proteins, amino acids and sulfhydryl compounds in foliar tissue of Arabidopsis thaliana during dark-induced and natural senescence. Plant Physiol. Biochem., 27: 203–209.
Desimone M., Wagner E., Johanningmeier U. 1998. Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis. Planta, 205: 459–466.
Dhindsa R. S., Plumb-Dhindsa P., Thorpe T. A. 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: 93–101.
Edreva A., Hadjiiska E. 1984. About the determination of sulfhydril (thiol) group content in plant material. Bulg. J. Plant Physiol. 10: 73–83 (in Bulg., English abstract).
Friedrich J.W., Huffaker R.C. 1980. Photosynthesis, leaf resistance and ribulose 1,5- bisphosphate carboxylase degradation in senescing barley leaves. Plant Physiol., 65: 1103–1107.
Gonzalez A., Steffen K. L., Lynch J. P. 1998. Light and excess manganese. Implications for oxidative stress in common bean. Plant Physiol. 118: 493–504.
Hodges D.M., Andrews Chr. J., Johnson D. A., Hamilton R.I, 1996. Antioxidant compound responses to chilling stress in differentially sensitive inbred maize lines. Physiol. Plantarum 98: 685–692.
Hörtensteiner S., Feller U. 2002. Nitrogen metabolism and remobilization during senescence. J. Exp. Bot., 53: 927–937.
Inada N., Sakai A., Kuroiwa H., Kuroiwa T. 1998. Three-dimensional analysis of the senescence program in rice (Oriza sativa L.) coleoptiles. Planta, 206: 585–597.
Irihimovitch V., Shapira M. 2000. Glutathione redox potential modulated by reactive oxygen species regulates translation of Rubisco large subunit in the chloroplast. J. Biol. Chem., 275 (21): 16289–95.
Jabs T. 1999. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem. Pharmac. 57: 231–245.
Kanazawa S., Sano S., Koshiba T., Ushimaru T. 2000. Changes in antioxidative enzymes in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence. Physiol. Plant., 109: 211–216.
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: 385–391.
Ku niak E., Skłodowska M. 2001. Ascorbate, glutathione and related enzymes in chloroplasts of tomato leaves infected by Botrytis cinerea. Plant Sci., 160: 723–731.
McKinney G. 1941. Absorbtion of light by chlorophyll solutions. J. Biol. Chem., 140: 315–322.
May M. J., Vernoux T., Leaver C., Van Montagu M., Inze D. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J. Exp. Bot., 49: 649–667.
McRae D.G., Thompson J.E. 1983. Senescence-dependent changes in superoxide anion production by illuminated chloroplasts from bean leaves. Planta, 158: 185–193.
Mehta R.A., Fawcett T.W., Porath D., Mattoo A. 1992. Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase/ oxygenase. J. Biol. Chem., 267: 2810–2816.
Merzlyak M.N., Hendry G.A.F. 1994. Free radical metabolism, pigment degradation and lipid peroxidation in leaves during senescence. Proc. Roy. Soc. Edinburg B 102: 459–471.
Minamikawa T., Toyooka K., Okamoto T., Hara-Nishimura I., Nishimura M. 2001. Degradation of ribulose-bisphosphate carboxylase by vacuolar enzymes of senescing French bean leaves: immunochemical and ultrastructural observations. Protoplasma 218: 144–153.
Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Sci., 7: 405–410.
Moreno J., Penarrubia L., Garcia-Ferris C. 1995. The mechanism of redox regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase turnover. A hypothesis. Plant Physiol. Biochem., 33: 121–127.
Munne-Bosch S., Jubany-Mari T., Alegre L. 2001. Drought-induced senescence is characterized by a loss of antioxidant defenses in chloroplasts. Plant Cell Environ., 24: 1319–1327.
Nakatani H.Y., Barber J. 1977. An improved method for isolating chloroplasts retaining their outer membranes. Biochim. Biophys. Acta, 461: 510–512.
Noctor G., Foyer, C.H. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol., 49: 249–279.
Pastori G. M., Del Rio L. A. 1997. Natural senescence of pea leaves. An activated oxygen-mediated function for peroxisomes. Plant Physiol., 113: 411–418.
Pe arrubia L, Moreno J. 1990. Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments. Arch. Biochem. Biophys. 281: 319–323.
Piquery L., Davoine C., Huault C., Billard J-P., 2000. Senescence of leaf sheaths of ryegrass stubble: changes in enzyme activities related to H2O2 metabolism. Plant Growth Regul., 30: 71–77.
Prochazkova D., Sairam R. K., Srivastava G. C., Singh D. V. 2001. Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Sci., 161: 765–771.
Reznick A.Z., Packer L. 1994. Oxidative damage to proteins: spectrophotometric method for carbonyl assay, In: Packer, L. (Ed.), Methods Enzymol., Academic Press, San Diego, CA, 233: 357–363.
Roulin S., Feller U. 1997. Light-induced proteolysis of stromal proteins in pea (Pisum sativum L.) chloroplasts: requirement for intact organelles. Plant Sci., 128: 31–41.
Roulin S., Feller U. 1998. Light independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations. Planta, 205: 297–304.
Simova-Stoilova L., Demirevska-Kepova K., Stoyanova Z. 2002. Ribulose-1,5-bisphosphate carboxylase/oxygenase specific proteolysis in barley chloroplasts during dark induced senescence. Photosynthetica, 40: 561–566.
Stadtman E. R. 1990. Covalent modification reactions are marking steps in protein turnover. Biochemistry, 29: 6323–6331.
Thompson J. E., Legge R.L., Barber R.F. 1987. The role of free radicals in senescence and wounding. New Phytol., 105: 317–344.
Van Hasselt P.R., 1972. Photo-oxidation of leaf pigments in Cucumus leaf discs during chilling. Acta Bot. Neerl., 21: 539–543.
Winter H., Robinson D. G., Heldt H. W., 1993. Subcellular volumes and metabolite concentrations in barley leaves. Planta, 191: 180–190.
Wittenbach V. A. 1978. Breakdown of ribulose bisphosphate carboxylase and changes in proteolytic activity during dark-induced senescence of wheat seedlings. Plant Physiol., 62: 604–608.
Wittenbach V. A. 1979. Ribulose bisphosphate carboxylase and proteolytic activity in wheat leaves from anthesis through senescence. Plant Physiol., 64: 884–887.
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Simova-Stoilova, L.P., Demirevska-Kepova, K.N. & Stoyanova, Z.P. Antioxidative protection in the leaves of dark-senescing intact barley seedlings. Acta Physiol Plant 27, 349–357 (2005). https://doi.org/10.1007/s11738-005-0011-9
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DOI: https://doi.org/10.1007/s11738-005-0011-9