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A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress

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Abstract

Intracellular proteases, together with molecular chaperones, are components of the cellular protein quality control system. Although the identity of chloroplast proteases has been revealed in recent years, little is known about their regulation. As a first step towards identifying unknown functional or regulatory components of the chloroplast proteolytic machinery, a genetic screen was devised with the aim of generating Arabidopsis thaliana (L.) Heynh. mutants impaired in chloroplast protein degradation. A streptomycin-resistance gene was fused to a cDNA construct that encodes an unstable mutant of the OE33 protein. This chimeric gene was used for transforming Arabidopsis plants. Analysis of transgenic plants revealed a correlation between streptomycin resistance and accumulation of the recombinant fusion protein. Seeds from transgenic plants that were sensitive to streptomycin were chemically mutagenized and screened for resistance to streptomycin. Such resistance could be due to stabilization of the protein caused by a mutation in the chloroplast proteolytic machinery. Genetic analysis of one of the mutants showed the mutation to be recessive, in a single nuclear gene. Further characterization of the mutant revealed that it was not a result of increased transcription of the transgene. Moreover, chloroplast lysates from the mutant plant showed decreased ATP-dependent degradation of a chloroplast protein substrate, consistent with the conclusion that the mutation impaired the proteolytic machinery. Physiological analysis revealed that upon cold stress, photoprotection and photosynthesis in the mutant was inhibited, suggesting that the chloroplast proteolytic machinery is involved in repairing damage incurred to the photosynthetic machinery upon exposure to cold temperatures.

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Abbreviations

NPQ:

non-photochemical quenching

OE33:

the 33-kDa subunit of the oxygen-evolving complex

RISP:

Rieske Fe–S protein

TR :

antibiotic-resistant transgenic plants

TS :

antibiotic-sensitive transgenic plants

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Acknowledgements

We thank Dr. Klaudia Brix for skillful help with confocal microscopy, Dr. Victor Raskin for excellent help and advice on chlorophyll fluorescence measurements, and Dr. Nir Ohad and Profs. Avi Levy and Itzhak Ohad for insightful discussions. This work was supported by grants from The Austrian Friends of The Hebrew University (to A.B. and Z.A.), the Israel Ministry of Science (to Z.A.), and the US–Israel Binational Agricultural Research and Development Fund—BARD (to Z.A.).

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Author notes

  1. Maggie Levy

    Present address: Department of Vegetable Crops, University of California, Davis, CA 95616, USA

  2. Andreas Bachmair

    Present address: Department of Developmental Biology of Plants, Max Planck Institute for Breeding Research, 50829, Cologne, Germany

Authors and Affiliations

  1. The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Faculty of Agriculture, The Hebrew University of Jerusalem, 76100, Rehovot, Israel

    Maggie Levy & Zach Adam

  2. Institute of Botany, University of Vienna, 1030, Vienna, Austria

    Andreas Bachmair

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Correspondence to Maggie Levy.

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Levy, M., Bachmair, A. & Adam, Z. A single recessive mutation in the proteolytic machinery of Arabidopsis chloroplasts impairs photoprotection and photosynthesis upon cold stress. Planta 218, 396–405 (2004). https://doi.org/10.1007/s00425-003-1120-6

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  • DOI: https://doi.org/10.1007/s00425-003-1120-6

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