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An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development

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Abstract

The ATP-dependent Clp protease has been well-characterized in Escherichia coli, but knowledge of its function in higher plants is limited. In bacteria, this two-component protease consists of a Ser-type endopeptidase ClpP, which relies on the ATP-dependent unfolding activity from an Hsp100 molecular chaperone to initiate protein degradation. In the chloroplasts of higher plants, multiple isoforms of the proteolytic subunit exist, with Arabidopsis having five ClpPs and four ClpP-like proteins termed ClpR predicted in its genome. In this work we characterized an Arabidopsis mutant impaired in one subunit of the chloroplast-localized Clp protease core, ClpR1. clpR1-1, a virescent mutant, carries a pre-mature stop codon in the clpR1 gene, resulting in no detectable ClpR1 protein. The accumulation of several chloroplast proteins, as well as most of the chloroplast-localized Clp protease subunits, is inhibited in clpR1-1. Unexpectedly, some plastid-encoded proteins do not accumulate, although their transcripts accumulate to wild-type levels. Maturation of 23S and 4.5S chloroplast ribosomal RNA (cp-rRNA) is delayed in clpR1-1, and both RNAs accumulate as higher molecular weight precursors. Also, chloroplasts in clpR1-1 are smaller than in wild type and have fewer thylakoid membranes with smaller grana stacks. We propose that a ClpR1-containing activity is required for chloroplast development and differentiation and in its absence both are delayed.

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Abbreviations

cp-rRNA:

Chloroplast ribosomal RNA

Lhcb2:

Light-harvesting Chl a/b binding protein

RbcL/LSU and RbcS/SSU:

Transcript and protein of large and Small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco)

PsaL, PsaD and PsaF:

Subunits XI, II and III of photosystem I (PS I)

Cpn60:

Chaperonin 60

β-ATPase:

CF1 β subunit of ATPase

PsbA and PsbD :

Transcripts encoding for D1 and D2 proteins of photosystem II (PSII)

NF:

Norflurazon

NPQ:

Non-photochemical excitation quenching

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Acknowledgement

This work was supported by the Howard Hughes Medical Institute and a grant from the Department of Energy to J.C., the Swedish Research Council for Environmental, Agricultural Sciences and Spatial Planning (Formas) to A.K.C., and an overseas postgraduate scholarship from the Natural Sciences and Engineering Research Council of Canada to T.M.S. S.K. was an EMBO long term fellow (ALTF 118–2000). J.C. is a Howard Hughes Medical Institute Investigator. We thank Takeshi Nakano (Riken Japan) for his help in analyzing chloroplast transcripts, Jason Lim for technical assistance and Olivier Loudet, Chris Schwartz and Jennifer Nemhauser for assisting in the map based cloning of clpR1-1. We thank Paul Sawchenko and the CCMI at Salk Institute for access to microscopy facilities. clpR1-2 seeds and the full length clpR1 cDNA clone were provided by The Arabidopsis Biological Resource Centre (ABRC), Ohio State University (Columbus, Ohio).

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  1. Shai Koussevitzky

    Present address: Department of Biochemistry and Molecular Biology, University of Nevada, Mail stop 200, Reno, NV, 89557-0014, USA

Authors and Affiliations

  1. Plant Biology Laboratory, The Salk Institute, La Jolla, CA, 92037, USA

    Shai Koussevitzky , Tony Giap & Joanne Chory

  2. Plant Molecular Biology, Botanical Institute, Göteborg University, Göteborg, Sweden

    Tara M. Stanne, Lars L. E. Sjögren & Adrian K. Clarke

  3. Laboratory of Neuronal Structure and Function, The Salk Institute, La Jolla, CA, USA

    Charles A. Peto

  4. Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA

    Yunde Zhao

  5. Howard Hughes Medical Institute, The Salk Institute, La Jolla, CA, USA

    Shai Koussevitzky  & Joanne Chory

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Correspondence to Shai Koussevitzky .

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Koussevitzky , S., Stanne, T.M., Peto, C.A. et al. An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development. Plant Mol Biol 63, 85–96 (2007). https://doi.org/10.1007/s11103-006-9074-2

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