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Tissue-specific and subcellular localization of phototropin determined by immuno-blotting

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Abstract

Phototropin (phot) is a UV/blue- light receptor mediating phototropic reactions of plants as a response to unilateral irradiation. Using an antiserum directed against the N-terminal part of Arabidopsis phot1, we show here cross-reaction with phototropin from Avena sativa, Eruca sativa, Glycine max, Lepidium sativum, Lycopersicon esculentum, Pisum sativum, Sinapis alba, and Zea mays. In all investigated plants, blue light irradiation led to a gel mobility shift of phototropin corresponding to an apparent increase in size of 2–3 kDa. This increase is transient: the apparent size of the phototropin band reverted back to the original size in the dark within 60–90 min. The capacity for in vitro phosphorylation increased to 350% (A. sativa) and 200% (L. sativum) at 90 min after a blue light pulse without an increase in the amount of phototropin protein. Starting from coleoptile tips of monocots that contained the highest concentration of phototropin, we found an exponential decrease in basipetal sections of equal size while a linear decrease was determined for dicots in basipetal sections starting from the section below the hypocotyl hook. We confirmed the membrane association of all phototropin in dark-grown seedlings; after a 2-min blue light pulse, however, 20% of phototropin was found in the cytosolic fraction and only 80% in the membrane fraction. Both fractions showed the gel mobility shift indicating light-dependent autophosphorylation. Detergent-free solubilization of phototropin with chaotropic reagents was investigated with etiolated A. sativa seedlings. Up to 95% of phototropin was solubilized with a mixture of sodium bromide and sodium diphosphate, and subsequently subjected to affinity purification using Cibachron Blue 3GA–agarose as a dinucleotide analogue. Immediately after solubilization, soluble phototropin still showed blue-light-dependent autophosphorylation but lost its activity within less than 1 h.

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Abbreviations

GFP :

Green fluorescent protein

phot :

Phototropin

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Acknowledgments

We thank Prof. W. R. Briggs and Dr. J. Christie, both at the Department of Plant Biology, Carnegie Institution, Stanford, California, for the antiserum directed against Arabidopsis phot1. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (Ru 108/31-4) and the Fonds der Chemischen Industrie, Frankfurt.

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Author notes

  1. Elke Knieb

    Present address: SiREEN AG, Am Klopferspitz 19a, 82152, Martinsried, Germany

  2. Michael Salomon

    Present address: Vertis Biotechnologie AG, Lise-Meitner Straße 30, 85354, Freising-Weihenstephan, Germany

Authors and Affiliations

  1. Department of Biology I (Botanik), Ludwig-Maximilians-Universität München, Menzingerstr. 67, 80638, München, Germany

    Elke Knieb, Michael Salomon & Wolfhart Rüdiger

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  1. Elke Knieb
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  2. Michael Salomon
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Correspondence to Wolfhart Rüdiger.

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Knieb, E., Salomon, M. & Rüdiger, W. Tissue-specific and subcellular localization of phototropin determined by immuno-blotting. Planta 218, 843–851 (2004). https://doi.org/10.1007/s00425-003-1164-7

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