Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module |
| |
Authors: | Yoshiaki Umemoto Toshiyoshi Araki |
| |
Institution: | (1) Laboratory for the Utilization of Aquatic Bioresources, Department of Life Science, Graduate School of Bioresources, Mie University, 1577 Kurimamachiya, Tsu Mie, 514-8507, Japan; |
| |
Abstract: | The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding
family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal
GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the
process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain
areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire
cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and
60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds
was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by
using a fluorescence-labeled CBM. |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|