Submitted to: Planta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/22/2003
Publication Date: 11/25/2003
Citation: Kim, H., Triplett, B.A., Pesacreta, T.C. 2004. Cotton-fiber germin-like protein II: Immunolocalization, purification and functional analysis. Planta. 218:525-535. Interpretive Summary: For most of the commercially important types of cotton there are two types of fibers: lint fibers that are used in yarn and fabric construction and the shorter fuzz fibers that are used in non-woven textiles and as substrates for manufacturing plastics and rayon. Our goal is to reduce the number of fuzz fibers and increase the number of lint fibers per seed. Both of these approaches will impact fiber quality and yield. Previously we isolated and characterized one gene that is actively read in normal cotton seeds and is not read in a mutant line where the product of fuzz fiber is blocked and development of lint fiber is restricted to one area of the seed. This newly identified cotton fiber gene is similar to several other genes previously characterized from other plants, however, there is much disagreement among plant scientists about the function. From this study we discovered that the gene is read only in fiber cells and not in other cotton tissues. Two approaches for discovering the function of this gene are reported in this manuscript. First, the protein coded by the gene was purified from developing cotton fibers and tested in activity assays for function. Three functions have been suggested in literature for this protein, however, the results from the activity assays indicate that the cotton protein does not function as any of three suggested enzymes. Secondly, an antibody was produced in rabbits against a very small region of the protein. This antibody was used to determine the precise location in the fiber cell where the protein accumulates. The protein was in the cytoplasm at very early stages and became localized in the cell wall at exactly the same time when fiber elongation reaches its maximum rate. This information will be used by researchers to isolate genetic information that codes for the promoter region of this gene so that new genetic information can be inserted only into cotton fiber cells. The data is also useful to researchers who are discovering the fundamental mechanisms for plant cell growth.
Technical Abstract: Germin-like protein appear to be ubiquitously distributed in the plant kingdom, however the function of these proteins in plants is not well understood. In several cereal species the "true" germins possess oxalate oxidease activity, however, none of the germin-like proteins are oxalate oxidates. Several other enzyme activities or functions have been suggested in the literature for germin-like protein: superoxide dismutase, ADP-glucose pyrophosphatase/phosphodiesterase, and auxin binding protein. Cotton (Gossypium hirsutum L.) contains a germin-like protein, GhGLP1 that shows tissue-specific accumulation in fiber. The fiber germin-like protein 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 to 14 days post-anthesis). During early phases of fiber development (2 to 4 days post anthesis), GhGLP1 locales to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7 to 10 days post-anthesis), GhGLP1 localizes to the apoplast. Cotton fiber extracts did not contain oxalate oxidase activity, nor did intact fibers stain for oxalate oxidase activity. A four-step purofication 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 to 14 days post-anthesis fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a germin-like protein accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.