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Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism

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Abstract

Using gene targeting by homologous recombination in Ceratodon purpureus, we were able to knock out four phytochrome photoreceptor genes independently and to analyze their function with respect to red light dependent phototropism, polarotropism, and chlorophyll content. The strongest phenotype was found in knock-out lines of a newly described phytochrome gene termed CpPHY4 lacking photo- and polarotropic responses at moderate fluence rates. Eliminating the atypical phytochrome gene CpPHY1, which is the only known phytochrome-like gene containing a putative C-terminal tyrosine kinase-like domain, affects red light-induced chlorophyll accumulation. This result was surprising, since no light dependent function was ever allocated to this unusual gene.

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Abbreviations

CTAB:

Cetyltrimethylammonium bromide

GT:

Gene targeting

HR:

Homologous recombination

ONP:

Oligonucleotide primer

RAcE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse transcription based polymerase chain reaction

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Acknowledgments

We would like to thank Dennis Hauser, Frankfurt a. M., Germany, for providing his software “MossBendingPlotter 3.0” visualizing the bending angles presented in Figs. 4, 5, 6, Tanja Gans for technical assistance and Man Mohan (Stowers Institute for medical research, Kansas City, MO, USA) for critical reading the manuscript and helpful discussion. The work was funded by grants of the Deutsche Forschungsgemeinschaft to CF (FO-290/4-1).

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  1. Sven Dienstbach

    Present address: Department of Genetics, Justus Liebig University, Heinrich-Buff Ring 58-62, 35392, Giessen, Germany

Authors and Affiliations

  1. Department of Plant Physiology, Justus Liebig University, Senckenbergstrasse 3, 35390, Giessen, Germany

    Franz Mittmann, Sven Dienstbach, Andrea Weisert & Christoph Forreiter

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  1. Franz Mittmann
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  2. Sven Dienstbach
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Correspondence to Christoph Forreiter.

Additional information

Accession number for CpPHY4: EU122393.

Electronic supplementary material

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425_2009_922_MOESM1_ESM.pdf

Schematic plot of the four different phytochrome genes and the corresponding knock-out constructs. Exon sequences are highlighted in orange. Restriction sites relevant for Southern-blot analysis are indicated above the gene. Red bars indicate size and position of the labeled PCR probe used for Southern-blot analysis. Inserted hygromycin and G418 resistance cassettes are visualized green. a CpPHY1 and cpphy1 knock-out; b CpPHY2 and cpphy2 knock-out; c CpPHY3 and cpphy3 knock-out; d CpPHY4 and cpphy4 knock-out, respectively (PDF 31 kb)

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Mittmann, F., Dienstbach, S., Weisert, A. et al. Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism. Planta 230, 27–37 (2009). https://doi.org/10.1007/s00425-009-0922-6

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