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Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii

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Abstract

The protein components of the NADP/thioredoxin system, NADP-thioredoxin reductase (NTR) and thioredoxin h, have been purified and characterized from the green alga, Chlamydomonas reinhardtii. The analysis of this system confirms that photoautotrophic Chlamydomonas cells resemble leaves in having both an NADP- and ferrodoxin-linked thioredoxin redox system. Chlamydomonas thioredoxin h, which is smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than thioredoxin m from the same source, cross-reacted with antisera to thioredoxin h from spinach (Spinacia oleracea L.) and wheat germ (Triticum vulgaris L.) but not with antisera to m or f thioredoxins. In these properties, the thioredoxin h resembled a thioredoxin from Chlamydomonas, designated Ch1, whose sequence was reported recently (P. Decottignies et al., 1991, Eur. J. Biochem. 198, 505–512). The differential reactivity of thioredoxin h with antisera was used to demonstrate that thioredoxin h is enriched outside the chloroplast. The NTR was purified from Chlamydomonas using thioredoxin h from the same source. Similar to its counterpart from other organisms, Chlamydomonas NTR had a subunit size of approx. 36 kDa and was specific for NADPH. Chlamydomonas NTR effectively reduced thioredoxin h from the same source but showed little activity with the other thioredoxins tested, including spinach thioredoxin h and Escherichia coli thioredoxin. Comparison of the reduction of Chlamydomonas thioredoxins m and h by each of the endogenous thioredoxin reductases, NTR and ferredoxin-thioredoxin reductase, revealed a differential specificity of each enzyme for thioredoxin. Thus, NTR showed increased activity with thioredoxin h and ferredoxin-thioredoxin reductase with thioredoxins m and f.

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Abbreviations

DEAE:

diethylaminoethyl

DTNB:

dithiobis(2-nitrobenzoic acid)

Fd:

ferredoxin

FTR:

ferredoxin-thioredoxin reductase

HPLC:

high-pressure liquid chromatography

Mr :

relative molecular mass

NADP-MDH:

NADP-malate dehydrogenase

NTR:

NADP-thioredoxin reductase

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  • Berstermann, A., Vogt, K., Follmann, H. (1983) Plant seeds contain several thioredoxins of regular size. Eur. J. Biochem. 131, 339–344

    Google Scholar 

  • Bruinsma, J. (1961) A comment on the spectrophotometric determination of chlorophyll. Biochim. Biophys. Acta 52, 576–578

    Google Scholar 

  • Bodenstein-Lang, J., Bush, A., Follmann, H. (1989) Animal and plant mitochondria contain specific thioredoxins. FEBS Lett. 258, 22–26

    Google Scholar 

  • Buchanan, B.B. (1980) Role of light in the regulation of chloroplast enzymes. Annu. Rev. Plant Physiol. 31, 341–374

    Google Scholar 

  • Crawford, N.A., Yee, B.C., Hutcheson, S.W., Wolosiuk, R.A., Buchanan, B.B. (1986) Enzyme regulation in C4 photosynthesis: purification, properties and activities of thioredoxins from C4 and C3 plants. Arch. Biochem. Biophys. 244, 1–15

    Google Scholar 

  • Crawford, N.A., Droux, M., Kosower, N.S., Buchanan, B.B. (1989) Evidence for the function of ferredoxin/thioredoxin system in the reductive activation of target enzymes of isolated intact chloroplasts. Arch. Biochem. Biophys. 271, 223–239

    Google Scholar 

  • Crétin, C., Luchetta, P., Joly, C., Decottignies, P., Lepiniec, L., Gadal, P., Sallanlin, M., Huet, J.-C., Pernollet, J.-C. (1990) Primary structure of sorghum malate dehydrogenase (NADP) deduced from cDNA sequence. Homology with malate dehydrogenase (NAD). Eur. J. Biochem. 192, 299–303

    Google Scholar 

  • Davies, D.R., Plaskitt, A. (1971) Genetic and structural analysis of cell wall formation in Chlamydomonas reinhardtii. Genet. Res. 17, 33–43

    Google Scholar 

  • Decottignies, P., Schmitter, J.-M., Jacquot, J.-P., Dutka, S., Picaud, A., Gadal, P. (1990) Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii. Arch. Biochem. Biophys. 280, 112–121

    Google Scholar 

  • Decottignies, P., Schmitter, J.-M., Dutka, S., Jacquot, J.-P., Miginiac-Maslow, M. (1991) Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. Eur. J. Biochem. 198, 505–512

    Google Scholar 

  • Droux, M., Miginiac-Maslow, M., Jacquot, J.P., Gadal, P., Crawford, N.A., Kosower, N.S., Buchanan, B.B. (1987) Ferredoxinthioredoxin reductase: a catalytically active dithiol group links photoreduced ferredoxin to thioredoxin functional in photosynthetic enzyme regulation. Arch. Biochem. Biophys. 256, 372–380

    Google Scholar 

  • Florencio, F.J., Yee, B.C., Johnson, T.C., Buchanan, B.B. (1988) An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach. Arch. Biochem. Biophys. 266, 496–507

    Google Scholar 

  • Gleason, F.K., Holmgren, A. (1988) Thioredoxin and related proteins in procaryotes. FEM Microbiol. Rev. 54, 271–298

    Google Scholar 

  • Hodges, M., Miginiac-Maslow, M., LeMaréchal, P., Rémy, R., (1990) The ATP-dependent post-translational modification of ferredoxin: NADP+ oxidase. Biochim. Biophys. Acta 1052, 446–452

    Google Scholar 

  • Holmgren, A. (1985) Thioredoxin. Annu. Rev. Biochem. 54, 237–271

    Google Scholar 

  • Huppe, H.C., Buchanan, B.B. (1989) Activation of a chloroplasttype of fructose bisphosphatase from Chlamydomonas reinhardtii by light-mediated agents. Z. Naturforsch. 44c, 487–494

    Google Scholar 

  • Huppe, H.C., de Lamotte-Guéry, F., Jacquot, J.-P., Buchanan, B.B. (1990) The ferredoxin-thioredoxin system of a green alga, Chlamydomonas reinhardtii. Identification and characterization of thioredoxins and ferredoxin-thioredoxin reductase components. Planta 180, 341–351

    Google Scholar 

  • Hutcheson, S.W., Buchanan, B.B. (1983) Enzyme regulation in crassulacean acid metabolism photosynthesis: studies on the ferredoxin/thioredoxin system of Kalanchoë daigremontiana. Plant Physiol. 72, 870–876

    Google Scholar 

  • Jacquot, J.-P., Vidal, J., Gadal, P., Schürmann, P. (1978) Evidence for the existence of several enzyme specific thioredoxins in plants. FEBS Lett. 96, 243–246

    Google Scholar 

  • Jacquot, J.-P., Buchanan, B.B., Martin, F., Vidal, J. (1981) Enzyme regulation in C4 photosynthesis. Purification and properties of thioredoxin-linked NADP-malate dehydrogenase from corn leaves. Plant Physiol. 68, 300–304

    Google Scholar 

  • Johnson, T.C., Cao, R.Q., Kung, J.E., Buchanan, B.B. (1987a) Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells. Planta 171, 321–331

    Google Scholar 

  • Johnson, T.C., Wada, K., Buchanan, B.B., Holmgren, A. (1987b) Reduction of purothionin by the wheat seed thioredoxin system. Plant Physiol 85, 446–451

    Google Scholar 

  • Kamo, M., Tsugita, A., Weissner, C., Wedel, N., Bartling, D., Hermenn, R.G., Aguilar, F., Garrdet-Salvi, L., Schürmann, P. (1989) Primary structure of spinach chloroplasts thioredoxin f: protein sequencing and analysis of complete cDNA clones for spinach chloroplast thioredoxin f. Eur. J. Biochem. 182, 315–322

    Google Scholar 

  • Langlotz, P., Wagner, W., Follmann, H. (1986) Green algae (Scenedesmus obliquus) contain three thioredoxins of regular size. Z. Naturforsch. 41c, 979–987

    Google Scholar 

  • Maeda, K., Tsugita, A., Dalzoppo, D., Vilbois, F., Schürmann, P. (1986) Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts. Eur. J. Biochem. 154, 197–203

    Google Scholar 

  • Marcus, F., Chamberlain, S.H., Chu, C., Masiarz, Shin, S., Yee, B.C., Buchanan, B.B. (1991) Plant thioredoxin h: an animal-like thioredoxin occurring in multiple cell compartments. Arch. Biochem. Biophys., in press

  • Mendiola-Morgenthaler, L., Leu, S., Boschetti, A. (1985) Isolation of biochemically active chloroplasts from Chlamydomonas. Plant Sci. 38, 33–39

    Google Scholar 

  • Miginiac-Maslow, M., Decottiginies, P., Jacquot, J.-P., Gadal, P. (1990) Regulation of corn leaf NADP-malate dehydrogenase light-activated by the photosynthetic electron flow. Effect of photoinhibition studied in a reconstituted system. Biochim. Biophys. Acta 1017, 273–279

    Google Scholar 

  • Shin, M., Tsujita, M., Tomizawa, H., Sakihama, N., Kamei, K., Oshino, R. (1990) Proteolytic degradation of ferredoxin-NADP reductase during purification from spinach. Arch. Biochem. Biophys. 279, 97–103

    Google Scholar 

  • Suske, G., Wagner, W., Follmann, H. (1979) NADP-dependent thioredoxin reductase and a new thioredoxin from wheat. Z. Naturforsch. 34c, 214–221

    Google Scholar 

  • Tsang, M. L.-S. (1981) Thioredoxin/glutaredoxin system of Chlorella. Plant Physiol. 68, 1098–1104

    Google Scholar 

  • Thelander, L. (1968) Studies on thioredoxin reductase from Escherichia coli B. Eur. J. Biochem. 4, 407–422

    Google Scholar 

  • Vidal, J., Gadal, P. (1983) Influence of light on phosphoenol pyruvate carboxylase in sorghum leaves. I. Identification and properties of two isoforms. Physiol. Plant. 57, 119–123

    Google Scholar 

  • Wolosiuk, R.A., Buchanan, B.B. (1977) Thioredoxin and glutathione regulate photosynthesis in chloroplasts. Nature 266, 565–567

    Google Scholar 

  • Wolosiuk, R.A., Crawford, N.A., Yee, B.C., Buchanan, B.B. (1979) Isolation of three thioredoxins from spinach leaves. J. Biol. Chem. 254, 1627–1632

    Google Scholar 

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Author notes

  1. Heather C. Huppe

    Present address: Department of Botany, University of British Columbia, V6T 1Z4, Vancouver, Canada

Authors and Affiliations

  1. Laboratoire de Physiologie Végétale Moléculaire, Université de Paris Sud, F-91405, Orsay Cedex, France

    Heather C. Huppe & Myroslawa Miginiac-Maslow

  2. Laboratoire de Biochimie Fonctionelle des Membranes Végétales, F-91198, Gif-sur-Yvette, France

    Alain Picaud

  3. Department of Plant Biology, University of California, 94720, Berkeley, CA, USA

    Bob B. Buchanan

Authors
  1. Heather C. Huppe
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  2. Alain Picaud
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  3. Bob B. Buchanan
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  4. Myroslawa Miginiac-Maslow
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Additional information

We thank Dr. P. Decottiginies for work on the HPLC, Dr. J.-P. Jacquot for help and discussion, Dr. M. Hodges for discussion and reading of the manuscript, Mme. Anne Trouabal for maintenance of our cell cultures and Dr. P. Gadal for providing facilities. H.C.H. was supported during this work by a Chateaubriand Fellowship. This work was supported in part by grant from the US-France Program of the National Science Foundation/Centre National de la Recerche Scientifique, and the National Aeronautics and Space Administration.

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Huppe, H.C., Picaud, A., Buchanan, B.B. et al. Identification of an NADP/thioredoxin system in Chlamydomonas reinhardtii . Planta 186, 115–121 (1991). https://doi.org/10.1007/BF00201506

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  • DOI: https://doi.org/10.1007/BF00201506

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