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Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids

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Abstract

The nucleic acid binding protein Whirly1 of barley has been located to both chloroplasts and the nucleus of the same cell. Immunogold labelling furthermore showed that in vivo Whirly1 does not strictly co-localize with DNA in chloroplasts, while it is closely associated with DNA in the nucleus. High-resolution imaging of Whirly1-GFP and PEND-RFP fusion proteins revealed that only a minor part of Whirly1 co-localizes with nucleoids. The co-localization with nucleoids is in accordance with the detection of Whirly1 in a conventionally prepared fraction of the transcriptionally active chromosome (TAC). By further purification and enrichment of transcriptional activity Whirly1, however, was lost from the TAC fraction. Knockdown of Whirly1 in transgenic barley plants had neither impact on transcription of selected protein coding genes nor on genes coding for ribosomal RNAs or tRNAs. The results of RIP-chip experiments showed that barley Whirly1 as its maize orthologue associates with a set of intron containing plastid RNAs. Taken together, the results suggest that plastid-located Whirly1 functions primarily in RNA metabolism rather than as a DNA binding protein.

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Abbreviations

TAC:

Transcriptionally active chromosome

RIP-chip:

RNA co-immunoprecipitation and chip hybridization

sRNAP:

Soluble RNA polymerase

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Acknowledgments

We thank Susanne Braun, Marita Beese, Cornelia Marthe and Reik Modrozynski for technical assistance. Christine Desel (CAU Kiel) is thanked for expert guidance in confocal laser scanning microscopy. Anke Schäfer is thanked for helpful ideas and Ying Miao (CAU Kiel) for critical reading of the manuscript and for help in preparation of the figures. Iris Meier (The Ohio State University, USA) is thanked for providing the antibody towards MFP1. We thank the Center of Biochemistry and Molecular Biology (CAU Kiel) for providing the phosphorimaging equipment. This work was supported by grants of the Deutsche Forschungsgemeinschaft (Kr1350/8, Kr1350/9) and an Emmy-Noether stipend to CSL.

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Authors and Affiliations

  1. Institute of Botany, Christian-Albrechts-University of Kiel, Olshausenstr. 40, 24098, Kiel, Germany

    Joanna Melonek, Evelyn Grabowski & Karin Krupinska

  2. Central Microscopy, Center of Biology, Christian-Albrechts-University of Kiel, Olshausenstr. 40, 24098, Kiel, Germany

    Maria Mulisch & Karin Krupinska

  3. Institute of Biology, Humboldt University of Berlin, Chausseestr. 117, 10115, Berlin, Germany

    Christian Schmitz-Linneweber

  4. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466, Gatersleben, Germany

    Götz Hensel

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  1. Joanna Melonek
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  2. Maria Mulisch
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  3. Christian Schmitz-Linneweber
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  4. Evelyn Grabowski
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  5. Götz Hensel
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  6. Karin Krupinska
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Correspondence to Karin Krupinska.

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Melonek, J., Mulisch, M., Schmitz-Linneweber, C. et al. Whirly1 in chloroplasts associates with intron containing RNAs and rarely co-localizes with nucleoids. Planta 232, 471–481 (2010). https://doi.org/10.1007/s00425-010-1183-0

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