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Planta

, Volume 193, Issue 2, pp 208–215 | Cite as

Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation

  • Carlos García-Ferris
  • Joaquín Moreno
Article

Abstract

When photoheterotrophic Euglena gracilis Z Pringsheim was subjected to nitrogen (N)-deprivation, the abundant photosynthetic enzyme ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was rapidly and selectively degraded. The breakdown began after a 4-h lag period and continued for a further 8 h at a steady rate. After 12 h of starvation, when the amount of Rubisco was reduced to 40%, the proteolysis of this enzyme slowed down while degradation of other proteins started at a similar pace. This resulted in a decline of culture growth, chloroplast disassembly — as witnessed by chlorophyll (Chl) loss — and cell bleaching. Experiments with spectinomycin, an inhibitor of chloroplastic translation, indicated that there was an absolute increase in the rate of Rubisco degradation in the N-deprived culture as compared with control conditions, where no significant carboxylase breakdown was detected. Oxidative aggregation of Rubisco (as detected by non-reductive electrophoresis) and association of the enzyme to membranes increased with time of N-starvation. Fluorescent labeling of oxidized cysteine (Cys) residues with monobromobimane indicated a progressive oxidation of Cys throughout the first hours of N-deprivation. It is concluded that Rubisco acts as an N store in Euglena, being first oxidized, and then degraded, during N-starvation. The mobilization of Rubisco allows sustained cell growth and division, at almost the same rate as the control (non-starved) culture, during 12 h of N-deprivation. Afterwards, breakdown is extended to other photosynthetic structures and the whole chloroplast is dismantled while cell growth is greatly reduced.

Key words

Chloroplast senescence Euglena Nitrogen storage Proteolysis Ribulose-1,5-bisphosphate carboxylase/oxygenase (oxidative modification) 

Abbreviations

Chl

chlorophyll

Cys

cysteine

Rubisco

ribulose-1,5-bisphosphate carboxylase/oxygenase

RuBP

ribulose-1,5-bisphosphate

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References

  1. Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lundqvist, T., Brändén, C.-I., Lorimer, G.H. (1989) Crystal structure of the active site of ribulose-bisphosphate carboxylase. Nature 337, 229–234Google Scholar
  2. Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24, 1–15Google Scholar
  3. Crafts-Brandner, S.J., Salvucci, M.E., Egli, D.E. (1990) Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5-phosphate kinase abundances and photosynthetic capacity during leaf senescence. Photosynth. Res. 23, 223–230Google Scholar
  4. Crawford, N.A., Droux, M., Kosower, N.S., Buchanan, B.B. (1989) Evidence for function of the ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts. Arch. Biochem. Biophys. 271, 223–239Google Scholar
  5. Douce, R., Joyard, J. (1982) Purification of the chloroplast envelope. In: Methods in chloroplast molecular biology, pp. 239–256, Edelman, M., Hallick, R.B., Chua, N.-H., eds. Elsevier Biomedical Press, AmsterdamGoogle Scholar
  6. Evans, J.R., Seemann, J.R. (1989) The allocation of protein nitrogen in the photosynthetic apparatus: cost, consequences, and control. In: Photosynthesis, pp. 183–205, Briggs, W.S., ed. Alan R. Liss. Inc., New YorkGoogle Scholar
  7. Faye, L., Chrispeels, M.J. (1987) Transport and processing of the glycosylated precursor of concanavalin A in jack-bean. Planta 170, 217–224Google Scholar
  8. Ferreira, R.B., Davies, D.D. (1987) Protein degradation in Lemna with particular reference to ribulose bisphosphate carboxylase. II. The effect of nutrient starvation. Plant Physiol. 83, 878–883Google Scholar
  9. Ferreira, R.B., Davies, D.D. (1989) Conversion of ribulose-1,5-bisphosphate carboxylase to an acidic and catalytically inactive form by extracts of osmotically stressed Lemna minor fronds. Planta 179, 448–455Google Scholar
  10. Ferreira, R.B., Shaw, N.M. (1989) Effect of osmotic stress on protein turnover in Lemna minor fronds. Planta 179, 456–465Google Scholar
  11. Field, C., Mooney, H.A. (1986) The photosynthesis-nitrogen relationship in wild plants. In: On the economy of plant form and function, pp. 25–35, Givinish, T.J., ed. Cambridge University Press, LondonGoogle Scholar
  12. García-Ferris, C., Moreno, J. (1993) Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase from Euglena gracilis. Photosynth. Res. 35, 55–66Google Scholar
  13. Goodwin, T.W. (1980) The biochemistry of carotenoids. Chapman and Hall, New YorkGoogle Scholar
  14. Huffaker, R.C. (1982) Biochemistry and physiology of leaf proteins. In: Encyclopedia of plant physiology, N.S., vol. 14A: Nucleic acids and proteins in plants, pp. 370–400, Boulter, D., Parthier, B., eds. Springer Verlag, BerlinGoogle Scholar
  15. Huner, N.P.A., Carter, J.V., Wold, F. (1982) Effects of reducing agent on the conformation of the isolated subunits of ribulose bisphosphate carboxylase/oxygenase from cold hardened and unhardened rye. Z. Pflanzenphysiol. 106, 69–80Google Scholar
  16. Kang, S.-M., Titus, J.S. (1980) Qualitative and quantitative change in nitrogenous compounds in senescing leaves and bark tissues of the apple. Physiol. Plant. 50, 285–290Google Scholar
  17. Kempner, E.S. (1982) Stimulation and inhibition of the metabolism and growth of Euglena gracilis. In: The biology of Euglena, vol. III, pp. 197–252, Buetow, D.E., ed. Academic Press, New YorkGoogle Scholar
  18. Knight, S., Anderson, I., Brändén, C.I. (1990) Crystallographic analysis of ribulose-1,5-bisphosphate carboxylase from spinach at 2.4 Å resolution: subunit interaction and active site. J. Mol. Biol. 215, 113–160Google Scholar
  19. Kosower, N.S., Kosower, E.M. (1987) Thiol labeling with bromobimanes. Methods Enzymol. 143, 76–84Google Scholar
  20. Kubawara, T., Hashimoto, Y. (1990) Purification of a dithiothreitolsensitive tetrameric protease from spinach PS II membranes. Plant Cell Physiol. 31, 581–589Google Scholar
  21. Landry, L.G., Pell, E.J. (1993) Modification of Rubisco and altered proteolytic activity in O3-stressed hybrid poplar (Populus maximowizii x trichocarpa). Plant Physiol. 101, 1355–1362Google Scholar
  22. Lorimer, G.H., Badger, M.R., Andrews, T.J. (1977) D-ribulose 1,5 bisphosphate carboxylase/oxygenase: improved methods for the activation and assay of catalytic activities. Anal. Biochem. 78, 68–75Google Scholar
  23. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275Google Scholar
  24. Mae, T., Makino, A., Ohira, K. (1983) Changes in the amounts of ribulose bisphosphate carboxylase synthesized and degraded during the life span of rice leaf (Oryza sativa L.). Plant Cell Physiol. 24, 1079–1086Google Scholar
  25. Mehta, R.A., Fawcett, T.W., Porath, D., Mattoo, A.K. (1992) Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase. J. Biol. Chem. 267, 2810–2816Google Scholar
  26. Moreno, J., García-Martinez, J.L. (1984) Nitrogen accumulation and mobilization in Citrus leaves throughout the annual cycle. Physiol. Plant. 61, 429–434Google Scholar
  27. Musgrove, J.E., Elderfield, P.D., Robinson, C. (1989) Endopeptidases in the stroma and thylakoids of pea chloroplasts. Plant Physiol. 90, 1616–1621Google Scholar
  28. Nettleton, A.M., Bhalla, P.L., Dalling, M.J. (1985) Characterization of peptide hydrolase activity associated with thylakoids of the primary leaves of wheat. J. Plant Physiol. 119, 35–43Google Scholar
  29. Ortiz, W., Wilson, C.J. (1988) Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis. Plant Physiol. 86, 554–561Google Scholar
  30. Peñarrubia, L., Moreno, J. (1990) Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments. Arch. Biochem. Biophys. 281, 319–323Google Scholar
  31. Peterson, L.W., Huffaker, R.C. (1975) Loss of RuDP carboxylase and increase of proteolytic activity in senescence on detached primary barley leaves. Plant Physiol. 78, 121–125Google Scholar
  32. Peterson, L.W., Kleinkopf, G.E., Huffaker, R.C. (1973) Evidence for lack of turnover of RuDP carboxylase in barley leaves. Plant Physiol. 51, 1042–1045Google Scholar
  33. Pringsheim, E.G., Pringsheim, O. (1952) Experimental elimination of chromatophores and eye-spot in Euglena gracilis. New Phytol. 51, 65–76Google Scholar
  34. Schmidt, A., Krämer, E. (1984) Membrane-bound cysteine oxidases in spinach, Chlorella, Synechococcus and Rhodopseudomonas. In: Advances in photosynthesis research, vol. III, pp. 525–528, Sybesma, C., ed. Martinus Nijhoff/Dr. W. Junk Publishers, The HagueGoogle Scholar
  35. Schmidt, G.W., Mishkind, M.L. (1983) Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts. Proc. Natl. Acad. Sci. USA 80, 2632–2636Google Scholar
  36. Shurtz-Swirski, R., Gepstein, S. (1985) Proteolysis of endogenous substrates in senescing oat leaves. Plant Physiol. 78, 121–125Google Scholar
  37. Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354Google Scholar
  38. Wittenbach, V.A. (1978) Breakdown of ribulose bisphosphate carboxylase and change in proteolytic activity during dark-induced senescence of wheat seedlings. Plant Physiol. 62, 604–608Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Carlos García-Ferris
    • 1
  • Joaquín Moreno
    • 1
  1. 1.Departament de Bioquímica i Biologia Molecular, Facultades de CienciasUniversitat de ValènciaBurjassotSpain

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