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Applied and Environmental Microbiology, May 2002, p. 2614-2618, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2614-2618.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Yutaka Tamaru,1,{dagger} Sadaharu Ui,1,{ddagger} Koichiro Murashima,1 Akihiko Kosugi,1 Helen Chan,1 Roy H. Doi,1* and Bo Liu2

Sections of Molecular and Cellular Biology,1 Plant Biology, University of California, Davis, California 956162

Received 15 October 2001/ Accepted 2 March 2002


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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.


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INTRODUCTION
 
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.


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Protoplast formation by crude culture supernatants from C. cellulovorans.
 
In preliminary experiments, cultured tobacco BY2 (TBY2) cells (14) were treated with the crude culture supernatant from C. cellulovorans (ATCC 35296) grown with glucose. To prepare the crude culture supernatant, C. cellulovorans was grown in 250 ml of medium containing 0.5% glucose by the method of Sleat et al. (17). The culture supernatant was concentrated and dialyzed as described previously (16) and dissolved in 10 ml of phosphate-buffered saline buffer (50 mM KH2PO4, 50 mM K2HPO4 [pH 6.8]). This solution was used as the crude culture supernatant and contained cellulosomes and noncellulosomal enzymes. Ten milliliters of cultured TBY2 cells was collected at 1,000 x g for 3 min. After removal of culture broth, the cells (2.5 x 106 to 3.0 x 106) were resuspended in 1 ml of the reaction mixture. The reaction mixture consisted of 250 µl of crude culture supernatant from C. cellulovorans, 250 µl of 0.2 M HEPES buffer (pH 7.0), and 500 µl of 0.8 M mannitol. Protoplast formation occurred in the dark at 40 rpm (New Brunswick Incubator Shaker, Model G25) and 22°C for 1 h as described previously (10). The number of released protoplasts was counted three times with a hemacytometer. When the treatment was done under acidic conditions (50 mM morpholineethanesulfonic acid [MES] buffer [pH 5.3]) for 1 h, only small amounts of protoplasts (0.3 x 105/ml) were released. On the other hand, many more protoplasts (1.5 x 106/ml) were released under neutral conditions (50 mM HEPES buffer [pH 7.0]) (Fig. 1A). These results indicated that neutral conditions were better than acidic conditions for the C. cellulovorans enzymes to form protoplasts. Thus, we chose neutral conditions for quantitative analysis of protoplast formation by crude culture supernatant from C. cellulovorans. Under neutral conditions, protoplasts were also released from the cultured Arabidopsis thaliana cells (Fig. 1B). The number of released protoplasts from the cultured TBY2 cells was designated the "protoplast formation activity."



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  		FIG. 1. Protoplast formation from cell culture suspensions. (A) Tobacco cells. (B) Arabidopsis thaliana cells. Left panels represent untreated cells. Right panels represent cells after treatment with crude supernatants from C. cellulovorans grown on pectin-containing medium.


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Effect of carbon sources in the media on protoplast formation and enzymatic activity.
 
We have observed that carbon sources in the medium have an effect on enzymatic activity of the crude culture supernatant and the subunit composition of the cellulosome (12, 19). Thus, in order to determine the effects of different carbon sources on the properties of the crude culture supernatant, C. cellulovorans was cultured with medium containing 0.5% glucose, cellobiose, xylan, locust bean gum (LBG), and pectin. Protoplast formation activity (Table 1), enzymatic activities (Table 1), amounts of protein (Table 1), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns (Fig. 2) were determined for each crude culture supernatant. Protein was measured by the method of Bradford (2) with a protein assay kit from Bio-Rad and with bovine serum albumin as a standard. The activities on carboxymethyl cellulase (CMC; medium viscosity, for CMCase), xylan (from birthwood, for xylanase), LBG (for mannanase), and pectin (from apple, for pectate lyase) were assayed at pH 6.0 and 37°C by measuring liberated reducing sugars as D-glucose equivalents by the Somogyi-Nelson method (22). The reaction mixture consisted of 250 µl of 1% substrate solution, 100 µl of 500 mM MES-NaOH buffer (pH 6.0), and 150 µl of enzyme solution. The incubation time was 30 min. Activity was expressed in units, with 1 U defined as the amount of enzyme releasing 1 µmol of reducing sugar per min.


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  		TABLE 1. Effect of carbon sources in culture media on protoplast formation and enzymatic activities of crude culture supernatants



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  		FIG. 2. SDS-PAGE analysis of the culture supernatants (A), the cellulose-binding fractions of crude culture supernatants (B), and Onozuka RS and commercial pectate lyase Y-23 (C). Lanes: 1, glucose; 2, cellobiose; 3, xylan; 4, LBG; 5, pectin. Numbers on the left are molecular masses of the markers. SDS-PAGE was performed with a 10% polyacrylamide gel by the method of Laemmli (8). The cellulose-binding fractions of crude culture supernatants were collected as described previously (16). Some proteins in the cellulose-binding fraction of crude culture supernatant from pectin-grown cells have been assigned to CbpA, EngE, EngK, ExgS, XynA, and ManA (B, lane 5) (13).

Interestingly, although the crude culture supernatant from LBG-grown cells showed enzymatic activities comparatively similar to those of other supernatants, the LBG supernatant did not show any protoplast formation activity. SDS-PAGE analyses of the LBG crude culture supernatant showed that there were no cellulose-binding proteins (Fig. 2B, lane 4). On the other hand, the total LBG supernatant contained many strong bands (Fig. 2A, lane 4). Our previous data showed that assembly into cellulosomes was necessary for the enzymatic subunits to bind to cellulose (12). Thus, these results suggested that the crude culture supernatant from LBG-grown cells formed just small amounts of cellulosome, if any. The fact that the crude culture supernatant from LBG did not show protoplast formation activity suggested that the assembly of cellulosomes was essential for protoplast formation activity.

Except for the crude culture supernatant from LBG-grown cells, the protoplast formation activities of the crude culture supernatants were almost proportional to the pectate lyase activities of the crude culture supernatants. These results suggested that the degradation of pectin was one of the rate-limiting steps for the crude culture supernatant from C. cellulovorans to produce protoplasts from cultured tobacco cells. Recently, we analyzed the enzymatic composition of the cellulosome from the crude culture supernatant from pectin-grown cells and showed that the cellulosome contained scaffolding protein CbpA, endoglucanase EngE, endoglucanase EngK, exoglucanase ExgS, xylanase XynA, and mannanase ManA (Fig. 2B, lane 5) (13). Although pectate lyase activity was detected in the crude culture supernatant from pectin-grown cells (Table 1), no pectate lyase enzymes were found in the cellulosomes (13). These results indicate that either pectin-grown cells produce a noncellulosomal pectate lyase enzyme or a cellulosomal pectate lyase was dissociated from the cellulosome during growth or during the isolation of cellulosomes.

Since the crude culture supernatant from pectin-grown cells showed the highest protoplast-forming activity (Table 1), this enzyme fraction was used for further experiments.


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Determination of contribution of cellulosomes to protoplast formation.
 
In order to confirm that cellulosomes are necessary for protoplast formation, the crude culture supernatant from pectin-grown cells was treated with cellulose and tested for protoplast formation. As described previously, cellulosomes could be removed from the crude culture supernatant by a cellulose binding treatment (16). The cellulose treatment of the crude culture supernatants from pectin-grown cells prevented protoplast formation completely (data not shown). These results strongly indicated that cellulosomes were essential for protoplast formation from cultured tobacco cells.

Previous data showed that anti-CbpA (formerly called anti-P170) inhibited the cellulose binding ability of cellulosomes, but not their CMCase activity (16). The crude culture supernatant from pectin-grown cells was pretreated with anti-CbpA and also tested for protoplast formation. The crude culture supernatant pretreated with anti-CbpA released no protoplasts from the tobacco cells. Since anti-CbpA treatment is considered to neutralize the cellulose binding ability of cellulosomes (16), these results indicated that binding of cellulosomes to cellulose in plant cell walls was an important process for protoplast formation.


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Comparison of protoplast formation activity of enzyme from C. cellulovorans with that of commercial enzymes.
 
Since the commercial cellulase Onozuka RS and the commercial pectate lyase Pectolyase Y-23 are widely used enzymes for protoplast formation, we compared the protoplast formation activities of the crude culture supernatant from pectin-grown cells with those of these commercial enzymes. The protein patterns of Onozuka RS and commercial pectate lyase Y-23 were determined by SDS-PAGE analysis (Fig. 2C). The commercial enzymes showed a simple protein pattern with three major bands, although lesser bands were also observed. The protein amounts of the commercial enzymes were 13% Onozuka RS powder and 0.95% Pectolyase Y-23, indicating that most of both enzyme powders are nonprotein components. For commercial enzymes, 0.8 M mannitol in 0.2 M MES buffer (pH 5.3) was used as the reaction solution as described previously (10). After cultured TBY 2 cells were treated with 10 mg of Onozuka RS per ml (the protein concentration is actually 1.3 mg/ml) for 1 h, no protoplasts were released. Then, the commercial pectate lyase Pectolyase Y-23 was added at 1 mg/ml (protein concentration, 0.0095 mg/ml). The combination of Onozuka RS and Pectolyase Y-23 released 8.5 x 105 protoplasts per ml (standard deviation = 0.8 x 105 protoplasts per ml) in 1 h from cultured TBY2 cells. The crude culture supernatant enzyme from pectin-grown C. cellulovorans cells (protein concentration in the reaction mixture, 0.20 mg/ml) released 15.7 x 105 protoplasts per ml in 1 h (Table 1). Based on the protein concentrations in the reaction mixtures, the crude culture supernatant from pectin-grown cells was considered to have a specific activity of protoplast formation higher than those of commercial enzyme solutions.

In addition to its higher activity, the crude culture supernatant from pectin-grown cells (A280 = 1.114) was optically cleaner than in the commercial enzyme solutions (A280 = 9.840) and had a clear watery appearance compared to the dark yellowish-brown color of the commercial enzymes. Since the major components of the commercial enzymes are nonprotein components as described above, the optical cleanness of the crude culture supernatant from pectin-grown cells might reflect a smaller amount of nonprotein components than those of commercial enzymes. Moreover, since the enzyme preparation was obtained only by ammonium sulfate precipitation of the crude culture supernatant and resuspension of the pellet with buffer, the enzyme was prepared readily. Therefore, the use of this crude culture supernatant should be useful to plant cell biologists and for any genetic, physiological, and commercial purposes requiring plant protoplasts.


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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.


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FOOTNOTES
 
* 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: rhdoi{at}ucdavis.edu. Back

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

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


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Applied and Environmental Microbiology, May 2002, p. 2614-2618, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2614-2618.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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