Skip to main content
SpringerLink
Log in

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

  • Published:
Planta Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

Chl:

chlorophyll

Cys:

cysteine

Rubisco:

ribulose-1,5-bisphosphate carboxylase/oxygenase

RuBP:

ribulose-1,5-bisphosphate

References

  • 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, 229–234

    Google Scholar 

  • Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidases in Beta vulgaris. Plant Physiol. 24, 1–15

    Google Scholar 

  • 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, 223–230

    Google Scholar 

  • 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, 223–239

    Google Scholar 

  • 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, Amsterdam

    Google Scholar 

  • 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 York

    Google Scholar 

  • Faye, L., Chrispeels, M.J. (1987) Transport and processing of the glycosylated precursor of concanavalin A in jack-bean. Planta 170, 217–224

    Google Scholar 

  • 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, 878–883

    Google Scholar 

  • 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, 448–455

    Google Scholar 

  • Ferreira, R.B., Shaw, N.M. (1989) Effect of osmotic stress on protein turnover in Lemna minor fronds. Planta 179, 456–465

    Google Scholar 

  • 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, London

    Google Scholar 

  • 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, 55–66

    Google Scholar 

  • Goodwin, T.W. (1980) The biochemistry of carotenoids. Chapman and Hall, New York

    Google Scholar 

  • 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, Berlin

    Google Scholar 

  • 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, 69–80

    Google Scholar 

  • 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, 285–290

    Google Scholar 

  • 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 York

    Google Scholar 

  • 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, 113–160

    Google Scholar 

  • Kosower, N.S., Kosower, E.M. (1987) Thiol labeling with bromobimanes. Methods Enzymol. 143, 76–84

    Google Scholar 

  • Kubawara, T., Hashimoto, Y. (1990) Purification of a dithiothreitolsensitive tetrameric protease from spinach PS II membranes. Plant Cell Physiol. 31, 581–589

    Google Scholar 

  • 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, 1355–1362

    Google Scholar 

  • 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, 68–75

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275

    Google Scholar 

  • 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, 1079–1086

    Google Scholar 

  • 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, 2810–2816

    Google Scholar 

  • Moreno, J., García-Martinez, J.L. (1984) Nitrogen accumulation and mobilization in Citrus leaves throughout the annual cycle. Physiol. Plant. 61, 429–434

    Google Scholar 

  • Musgrove, J.E., Elderfield, P.D., Robinson, C. (1989) Endopeptidases in the stroma and thylakoids of pea chloroplasts. Plant Physiol. 90, 1616–1621

    Google Scholar 

  • 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, 35–43

    Google Scholar 

  • Ortiz, W., Wilson, C.J. (1988) Induced changes in chloroplast protein accumulation during heat bleaching in Euglena gracilis. Plant Physiol. 86, 554–561

    Google Scholar 

  • 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, 319–323

    Google Scholar 

  • 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, 121–125

    Google Scholar 

  • Peterson, L.W., Kleinkopf, G.E., Huffaker, R.C. (1973) Evidence for lack of turnover of RuDP carboxylase in barley leaves. Plant Physiol. 51, 1042–1045

    Google Scholar 

  • Pringsheim, E.G., Pringsheim, O. (1952) Experimental elimination of chromatophores and eye-spot in Euglena gracilis. New Phytol. 51, 65–76

    Google Scholar 

  • 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 Hague

    Google Scholar 

  • 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, 2632–2636

    Google Scholar 

  • Shurtz-Swirski, R., Gepstein, S. (1985) Proteolysis of endogenous substrates in senescing oat leaves. Plant Physiol. 78, 121–125

    Google Scholar 

  • 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, 4350–4354

    Google Scholar 

  • Wittenbach, V.A. (1978) Breakdown of ribulose bisphosphate carboxylase and change in proteolytic activity during dark-induced senescence of wheat seedlings. Plant Physiol. 62, 604–608

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departament de Bioquímica i Biologia Molecular, Facultades de Ciencias, Universitat de València, Av. Dr. Moliner 50, E-46100, Burjassot, Spain

    Carlos García-Ferris & Joaquín Moreno

Authors
  1. Carlos García-Ferris
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joaquín Moreno
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

We thank Drs. Pablo Vera and Ismael Rodrigo (Univ. Politécnica, Valencia, Spain) for advice and facilities in raising and collecting the anti-Rubisco serum. This work was supported by grants PB87-0353 and PB92-0821 of DGICYT and by a fellowship of the Spanish Ministerio de Educación y Ciencia (awarded to C.G.-F.).

Rights and permissions

Reprints and permissions

About this article

Cite this article

García-Ferris, C., Moreno, J. Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation. Planta 193, 208–215 (1994). https://doi.org/10.1007/BF00192532

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00192532

Key words

Search

Navigation