Formation of Protoplasts from Cultured Tobacco Cells and <em>Arabidopsis thaliana</em> by the Action of Cellulosomes and Pectate Lyase from <em>Clostridium cellulovorans</em>

Yutaka Tamaru

Sadaharu Ui

Koichiro Murashima

Akihiko Kosugi

Helen Chan

Roy Doi

Bo Liu

Sections of Molecular and Cellular Biology,^{Plant Biology, University of California, Davis, California 95616}²

^{Corresponding author. Mailing address: Sections of Molecular and Cellular Biology, University of California, Davis, CA 95616. Phone: (530) 752-3191. Fax: (530) 752-3085. E-mail:}ude.sivadcu@iodhr.

^{Present address: Faculty of Bioresources, Mie University, Tsu 514-8507, Japan.}

^{Present address: Faculty of Engineering, Yamanashi University, Kofu 400-8511, Japan.}

Received 2001 Oct 15; Accepted 2002 Mar 2.

Abstract

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.

Abstract

Clostridium cellulovorans produces an extracellular enzyme complex (cellulosome) containing a variety of cellulolytic subunits attached to the nonenzymatic scaffolding component termed CbpA (3). Furthermore, C. cellulovorans produces noncellulosomal cellulolytic enzymes, such as EngD (5) and EngF (15). Our previous data showed that the cellulosome was an essential enzyme complex for the degradation of crystalline cellulose (16). The role, if any, of noncellulosomal cellulases for degradation of crystalline cellulose is still unknown. So far, we have cloned and sequenced eight cellulosomal cellulase genes from C. cellulovorans: engB (4), engE (18), engH (20), engK (20), engL (19), engM (19), engY (21), and exgS (11). In addition, the genes coding for cellulosomal mannanase (manA) (19), pectate lyase (pelA) (21), and xylanase (xynA) (6; A. Kosugi, K. Murashima, and R. H. Doi, submitted for publication) have been cloned and sequenced. Since plant cell walls contain hemicellulose and pectin as well as cellulose (9), the existence of cellulosomal hemicellulases and pectate lyase suggests that the C. cellulovorans cellulosome can degrade plant cell walls effectively.

Protoplast formation from plant tissues is an important step in plant biotechnology, since protoplasts are used as materials for cell fusion and transformation. Although enzymatic isolation of plant protoplasts is an established laboratory procedure, conditions for isolation of protoplasts must be optimized for each tissue (1). Furthermore, impurities in commercial enzyme may damage the isolated protoplasts and decrease their viability (7). Thus, development of a highly pure enzyme preparation for protoplast isolation is important to obtain quality protoplasts.

In this study, we determined the protoplast formation ability of enzymes from C. cellulovorans. We demonstrate that these enzymes release protoplasts from cultured tobacco cells and have a higher activity of protoplast formation than commercial enzymes based on total protein in the enzyme preparations. The essential contribution of cellulosomes to protoplast formation is also described.

Acknowledgments

We thank J. Lee and M. Lai for assistance with plant cell cultures.

This research was supported in part by grant DE-FG03-92ER20069 from the U.S. Department of Energy.

Acknowledgments

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