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The maize brittle1 gene encodes amyloplast membrane polypeptides

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Abstract

A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies. Following affinity purification, the antibodies recognized a set of 38-to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm. Bt1 proteins were not detected with the preimmune antibodies. A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP. At the same time, the ratio of slow to fast-migrating forms of the protein decreased. During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes. They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery. These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins.

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Abbreviations

AACP:

ATP/ADP carrier protein

DAP:

days after pollination

PVDF:

polyvinylidine difluoride

GST:

glutathione-sulfur-transferase

EM:

electron microscope

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Authors and Affiliations

  1. Laboratory of Genetics, University of Wisconsin, 53706, Madison, WI, USA

    Thomas D. Sullivan

  2. Department of Regulation Biology, Saitama University, 338, Urawa, Japan

    Yasuko Kaneko

Authors
  1. Thomas D. Sullivan
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  2. Yasuko Kaneko
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Additional information

This research was supported in part by a NRI Competitive Grants Program/USDA grant, No. 91-37301-6380. This is Laboratory of Genetics paper number 3395. We would like to thank Oliver Nelson for field space, genetic stocks and for helpful discussions of the experiments and this manuscript, and Ron Okagaki for discusions of this work and manuscript. Patrick Masson has graciously provided laboratory space, as well as helpful discussions of the experiments and the manuscript. Field assistance was provided by Wayne Smith, Dave Heller, Anne Bertinuson and Vien Pham, who also provided technical assistance in the laboratory. We also thank Dave Hondred for technical discussions concerning protein blotting, Charles Boyer for providing sh2 and bt2 maize stocks, and Walter Neupert and associates for providing the anti-AACP antibodies.

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Sullivan, T.D., Kaneko, Y. The maize brittle1 gene encodes amyloplast membrane polypeptides. Planta 196, 477–484 (1995). https://doi.org/10.1007/BF00203647

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  • DOI: https://doi.org/10.1007/BF00203647

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