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Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development

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Abstract

70 kDa heat shock proteins (Hsp70s) act as molecular chaperones involved in essential cellular processes such as protein folding and protein transport across membranes. They also play a role in the cell’s response to a wide range of stress conditions. The Arabidopsis family of Hsp70s homologues includes two highly conserved proteins, cpHsc70-1 and cpHsc70-2 which are both imported into chloroplasts (Su and Li in Plant Physiol 146:1231–1241, 2008). Here, we demonstrate that YFP-fusion proteins of both cpHsc70-1 and cpHsc70-2 are predominantly stromal, though low levels were detected in the thylakoid membrane. Both genes are ubiquitously expressed at high levels in both seedlings and adult plants. We further show that both cpHsc70-1 and cpHsc70-2 harbour ATPase activity which is essential for Hsp70 chaperone activity. A previously described T-DNA insertion line for cpHsc70-1 (ΔcpHsc70-1) has variegated cotyledons, malformed leaves, growth retardation, impaired root growth and sensitivity to heat shock treatment. In addition, under stress conditions, this mutant also exhibits unusual sepals, and malformed flowers and sucrose concentrations as low as 1% significantly impair growth. cpHsc70-1/cpHsc70-2 double-mutants are lethal. However, we demonstrate through co-suppression and artificial microRNA (amiRNA) approaches that transgenic plants with severely reduced levels of both genes have a white and stunted phenotype. Interestingly, chloroplasts in these plants have an unusual morphology and contain few or no thylakoid membranes. Our data show that cpHsc70-1 and cpHsc70-2 are essential ATPases, have overlapping roles and are required for normal plastid structure.

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Abbreviations

YFP:

Yellow fluorescent protein

ER:

Endoplasmic reticulum

Hsp70:

70 kDa heat shock proteins

amiRNA:

Artificial microRNA

VIPP1:

Vesicle inducing protein in plastids 1

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Acknowledgments

We thank Natalie Allcock and Stefan Hyman for the electron microscopy, NASC for providing the seeds for the T-DNA insertion lines, Plant systems Biology at VIB-Ghent University for providing the vector pB7YWG2 and Mark Curtis at the University of Zurich for the pMDC vectors. We also thank Poul Eric Jensen (University of Copenhagen, Denmark) for providing PsaF and PsaD antibodies. This work was financed by a grant from The Norwegian Research Council (178071) to Simon Geir Møller.

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Correspondence to Simon Geir Møller.

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M. Latijnhouwers and X.-M. Xu contributed equally to this work.

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425_2010_1192_MOESM1_ESM.tif

Schematic diagram representing genomic sequences of cpHsc70-1 and cpHsc70-2. Lines represent untranslated regions and boxes represent exons. The locations of the T-DNA insertions SALK_140810 and GK544B06 are indicated. Primers annealing sites are indicated with arrows. The primer sets LP_140810/RP_140810 and LP_95259/RP_95259 were used to genotype individual plants (Fig 3a). The sets 70-1_F3/70-1_R3 and 70-2_F3/70-2_R3 were used in RT-PCR (Fig 3b).(TIFF 954 kb)

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Latijnhouwers, M., Xu, XM. & Møller, S.G. Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development. Planta 232, 567–578 (2010). https://doi.org/10.1007/s00425-010-1192-z

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