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.

Methodology Characterization of a rice germin-like protein gene promoter

“…In a detailed study of the in planta role of OXO in Lolium perenne, Le Deunff et al (2004) concluded that germin OXOs might be instrumental during critical events in the life of plants, such as cutting and senescence by initiating H 2 O 2 -mediated defenses against pathogens and foraging animals. Other mechanisms such as reinforcement of cell walls by GLPs have also been suggested (Lane, 1994;Christensen et al, 2000;Kim et al, 2004). The evidence of possible promoter induction by wounding in the present study may also strengthen the hypothesis that GLPs play an important role in generalized plant defense (Schafleitner and Wilhelm, 2002;Segarra et al, 2003).…”

A germin-like protein gene of rice increased superoxide dismutase activity in transformed tobacco

“…The absence of any OXO activity in leaves, stems, and roots of the transgenic tobacco overexpressing OsRGLP1 is in accordance with a previous finding (Kim et al 2004) concerning another GLP. Similarly, a GLP isolated from cells of Barbula unguiculata is connected with MnSOD activity but not with OXO activity (Yamahara et al 1999).…”

Cotton fiber germin-like protein. I. Molecular cloning and gene expression

“…Collectively, these results demonstrate that sequence similarity is insufficient evidence to predict accurately how GLPs function in plant cells. Purification and characterization of the GhGLP1 protein is the next step for establishing how this gene product functions in elongating cotton fibers (Kim et al 2003).…”

“…However, this proposal is questionable because several groups have been unable to show Mn-SOD activity in their purified germin preparations (Carter and Thornburg 2000;Bernier and Berna 2001;Whittaker and Whittaker 2002;Kim et al 2003;S. Bornemann, personal communication;B.G.…”

“…Although widely distributed in the plant kingdom, only one GLP, that of Silene vulgaris (Bringezu et al 1999), has been reported to have OxO activity. The remaining GLPs that have been tested do not show OxO activity Ohmiya 2002;Ohmiya et al 1998;Wei et al 1998;Yamahara et al 1999;Carter and Thornburg 2000;Kim et al 2003).…”