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

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.