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Cotton-fiber germin-like protein. II: Immunolocalization, purification, and functional analysis

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Abstract

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.

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Abbreviations

ABP:

Auxin binding protein

AGPPase:

ADP-Glucose pyrophosphatase/phosphodiesterase

bis-PNPP:

Bis-p-nitrophenol phosphate

ConA:

Concanavalin A

DOA:

Day of anthesis

DPA:

Days post-anthesis

GLP:

Germin-like protein

Mn-SOD:

Manganese superoxide dismutase

OxO:

Oxalate oxidase

PBS:

Phosphate-buffered saline

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Acknowledgements

We thank Chris Hawes for generously providing the JIM 84 antibody and Professors Byron Lane, François Bernier, and Robert Thornburg for critically reviewing the manuscript. The Louisiana State Support Program of Cotton Incorporated and United States Department of Agriculture, Agricultural Research Service (CRIS 6435-21440-002-00D) provided support for this project. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA.

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  1. Southern Regional Research Center, USDA, ARS, New Orleans, LA 70124, USA

    Hee Jin Kim & Barbara A. Triplett

  2. The Microscopy Center, University of Louisiana, Lafayette, LA 70504, USA

    Thomas C. Pesacreta

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  1. Hee Jin Kim
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Correspondence to Barbara A. Triplett.

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Kim, H.J., Pesacreta, T.C. & Triplett, B.A. Cotton-fiber germin-like protein. II: Immunolocalization, purification, and functional analysis. Planta 218, 525–535 (2004). https://doi.org/10.1007/s00425-003-1134-0

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