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

Planta volume 232, pages 567–578 (2010)Cite this article

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

References

  • Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

    Article  PubMed  Google Scholar 

  • Aseeva E, Ossenbühl F, Sippel C, Cho WK, Stein B, Eichacker LA, Meurer J, Wanner G, Westhoff P, Soll J, Vothknecht UC (2007) Vipp1 is required for basic thylakoid membrane formation but not for the assembly of thylakoid protein complexes. Plant Physiol Biochem 45:119–128

    Article  CAS  PubMed  Google Scholar 

  • Brodsky JL (1996) Post-translational protein translocation: not all hsc70s are created equal. Trends Biochem Sci 21:122–126

    CAS  PubMed  Google Scholar 

  • Brown CM, MacKinnon JD, Cockshutt AM, Villareal TA, Campbell DA (2008) Flux capacities and acclimation costs in Trichodesmium from the Gulf of Mexico. Mar Biol 154:413–422

    Article  Google Scholar 

  • Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high throughput functional analysis of genes in planta. Plant Physiol 133:462–469

    Article  CAS  PubMed  Google Scholar 

  • Froehlich JE, Wilkerson CG, Ray WK, McAndrew RS, Osteryoung KW, Gage DA, Phinney BS (2003) Proteomic study of the Arabidopsis thaliana chloroplastic envelope membrane utilizing alternatives to traditional two-dimensional electrophoresis. J Proteome Res 2:413–425

    Article  PubMed  Google Scholar 

  • Gutensohn M, Fan E, Frielingsdorf S, Hanner P, Hou B, Hust B, Klösgen RB (2006) Toc, Tic, Tat et al.: structure and function of protein transport machineries in chloroplasts. J Plant Physiol 163:333–347

    Article  CAS  PubMed  Google Scholar 

  • Guy CL, Li QB (1998) The organization and evolution of the spinach stress 70 molecular chaperone gene family. Plant Cell 10:539–556

    Article  CAS  PubMed  Google Scholar 

  • Karimi M, De Meyer B, Hilson P (2005) Modular cloning in plant cells. Trends Plant Sci 10:103–105

    CAS  PubMed  Google Scholar 

  • Kourtz L, Ko K (1997) The early stage of chloroplast protein import involves Com70. J Biol Chem 272:2808–2813

    Article  CAS  PubMed  Google Scholar 

  • Kroll D, Meierhoff K, Bechtold N, Kinoshita M, Westphal S, Vothknecht UC, Soll J, Westhoff P (2001) VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation. Proc Natl Acad Sci USA 98:4238–4242

    Article  CAS  PubMed  Google Scholar 

  • Latijnhouwers M, Hawes C, Carvalho C, Oparka K, Gillingham AK, Boevink P (2005) An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1. Plant J 44:459–470

    Article  CAS  PubMed  Google Scholar 

  • Liu C, Willmund F, Whitelegge JP, Hawat S, Knapp B, Lodha M, Schroda M (2005) J-domain protein CDJ2 and HSP70B are a plastidic chaperone pair that interacts with vesicle-inducing protein in plastids 1. Mol Biol Cell 16:1165–1177

    Article  CAS  PubMed  Google Scholar 

  • Liu C, Willmund F, Golecki JR, Cacace S, Hess B, Markert C, Schroda M (2007) The chloroplast HSP70B-CDJ2-CGE1 chaperones catalyse assembly and disassembly of VIPP1 oligomers in Chlamydomonas. Plant J 50:265–277

    Article  CAS  PubMed  Google Scholar 

  • Madueno F, Napier JA, Gray JC (1993) Newly imported rieske iron-sulfur protein associates with both Cpn60 and Hsp70 in the chloroplast stroma. Plant Cell 5:1865–1876

    Article  CAS  PubMed  Google Scholar 

  • Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62:670–684

    Article  CAS  PubMed  Google Scholar 

  • Miernyk JA (1997) The 70 kDa stress-related proteins as molecular chaperones. Trends Plant Sci 2:180–187

    Article  Google Scholar 

  • Nielsen E, Akita M, Davila-Aponte J, Keegstra K (1997) Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone. EMBO J 16:935–946

    Article  CAS  PubMed  Google Scholar 

  • Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690

    Article  CAS  PubMed  Google Scholar 

  • Peltier JB, Cai Y, Sun Q, Zabrouskov V, Giacomelli L, Rudella A, Ytterberg AJ, Rutschow H, van Wijk KJ (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol Cell Proteomics 5:114–133

  • Ratnayake RM, Inoue H, Nonami H, Akita M (2008) Alternative processing of Arabidopsis Hsp70 precursors during protein import into chloroplasts. Biosci Biotechnol Biochem 72:2926–2935

    Article  CAS  PubMed  Google Scholar 

  • Rial DV, Arakaki AK, Ceccarelli EA (2000) Interaction of the targeting sequence of the chloroplast precursors with Hsp70 molecular chaperones. Eur J Biochem 267:6239–6248

    Article  CAS  PubMed  Google Scholar 

  • Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K, Weisshaar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53:247–259

    Article  CAS  PubMed  Google Scholar 

  • Rutschow H, Ytterberg AJ, Friso G, Nilsson R, van Wijk KJ (2008) Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis. Plant Physiol 48:156–175

    Article  Google Scholar 

  • Schnell DJ, Kessler F, Blobel G (1994) Isolation of components of the chloroplast protein import machinery. Science 266:1007–1012

    Article  CAS  PubMed  Google Scholar 

  • Schroda M, Vallon O, Wollman FA, Beck CF (1999) A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition. Plant Cell 11:1165–1178

    Article  CAS  PubMed  Google Scholar 

  • Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133

    Article  CAS  PubMed  Google Scholar 

  • Seigneurin-Berny D, Salvi D, Joyard J, Rolland N (2008) Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation. Curr Protoc Cell Biol Chapter 3:Unit 3.30

  • Shi LX, Theg SM (2010) A stromal heat shock protein 70 system functions in protein import into chloroplasts in the moss Physcomitrella patens. Plant Cell 22:205–220

    Article  CAS  PubMed  Google Scholar 

  • Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Exp Purif 41:207–234

    Article  CAS  Google Scholar 

  • Su PH, Li HM (2008) Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds. Plant Physiol 146:1231–1241

    Article  CAS  PubMed  Google Scholar 

  • Sung DY, Kaplan F, Guy CL (2001a) Plant Hsp70 molecular chaperones: protein structure, gene family, expression and function. Physiol Plant 113:443–451

    Article  CAS  Google Scholar 

  • Sung DY, Vierling E, Guy CL (2001b) Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol 126:789–800

    Article  CAS  PubMed  Google Scholar 

  • Toufighi K, Brady SM, Austin R, Ly E, Provart NJ (2005) The botany array resource: e-northerns, expression angling, and promoter analyses. Plant J 43:153–163

    Article  CAS  PubMed  Google Scholar 

  • Tsugeki R, Nishimura M (1993) Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP+ reductase upon its import into chloroplasts. FEBS Lett 320:198–202

    Article  CAS  PubMed  Google Scholar 

  • Wimmer B, Lottspeich F, Van der Kle I, Veenhuts M, Gietl C (1997) The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene. Proc Natl Acad Sci USA 94:13624–13629

    Article  CAS  PubMed  Google Scholar 

  • Yalovsky S, Paulsen H, Michaeli D, Chitnis PR, Nechushtai R (1992) Involvement of a chloroplast HSP70 heat shock protein in the integration of a protein (light-harvesting complex protein precursor) into the thylakoid membrane. Proc Natl Acad Sci USA 89:5616–5619

    Article  CAS  PubMed  Google Scholar 

  • Yokthongwattana K, Chrost B, Behrman S, Casper-Lindley C, Melis A (2001) Photosystem II damage and repair cycle in the green alga Dunaliella salina: involvement of a chloroplast-localized HSP70. Plant Cell Physiol 42:1389–1397

    Article  CAS  PubMed  Google Scholar 

  • Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632

    Article  CAS  PubMed  Google Scholar 

  • Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ (2008) Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS One 3:e1994

    Google Scholar 

  • Zybailov B, Sun Q, van Wijk KJ (2009a) Workflow for large scale detection and validation of peptide modifications by RPLC-LTQ-Orbitrap: application to the Arabidopsis thaliana leaf proteome and an online modified peptide library. Anal Chem 81:8015–8024

    Article  CAS  PubMed  Google Scholar 

  • Zybailov B, Friso G, Kim J, Rudella A, Rodríguez VR, Asakura Y, Sun Q, van Wijk KJ (2009b) Large scale comparative proteomics of a chloroplast Clp protease mutant reveals folding stress, altered protein homeostasis, and feedback regulation of metabolism. Mol Cell Proteomics 8:1789–1810

    Article  CAS  PubMed  Google Scholar 

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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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Affiliations

  1. Centre of Organelle Research, Faculty of Science and Technology, University of Stavanger, 4021, Stavanger, Norway

    Maita Latijnhouwers, Xiang-Ming Xu & Simon Geir Møller

  2. Norwegian Centre for Movement Disorders, Stavanger University Hospital, 4068, Stavanger, Norway

    Simon Geir Møller

Authors
  1. Maita Latijnhouwers
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  2. Xiang-Ming Xu
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  3. Simon Geir Møller
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Corresponding author

Correspondence to Simon Geir Møller.

Additional information

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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Keywords

  • Artificial microRNA
  • ATPase
  • Chloroplast
  • Hsp70
  • Photosynthesis
  • Stroma
  • Thylakoid