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Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

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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.

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Acknowledgments

We thank S. Karita from Mie University for the valuable information provided for this paper. This study was supported by a Grant-in-Aid for Scientific Research (C; no. 19580235, 2007-2008) by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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  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

    Yoshiaki Umemoto & Toshiyoshi Araki

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  1. Yoshiaki Umemoto
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  2. Toshiyoshi Araki
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Correspondence to Toshiyoshi Araki.

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Umemoto, Y., Araki, T. Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module. Mar Biotechnol 12, 24–31 (2010). https://doi.org/10.1007/s10126-009-9196-z

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