Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9353-9357, September 1995 Plant Biology A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants YEHUDIT AMOR*, CANDACE H. HAIGLERt, SARAH JOHNSONt, MELODY WAINSCOTTt, AND DEBORAH P. DELMER*t *Department of Botany, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel; and tDepartment of Biological Sciences, Texas Tech University, Lubbock, TX 79409 Communicated by Joseph E. Varner§, Washington University, St. Louis, MO, June 22 1995 ABSTRACT Sucrose synthase (SuSy; EC 2.4.1.13; sucrose starch deposition and extensive cell wall degeneration in + UDP = UDPglucose + fructose) has always been studied mutant maize endosperm deficient in SuSy activity (11). as a cytoplasmic enzyme in plant cells where it serves to Developing cotton fibers transiently synthesize callose at the degrade sucrose and provide carbon for respiration and onset of secondary wall deposition followed by massive syn- synthesis of cell wall polysaccharides and starch. We report thesis of cellulose, making them an excellent system for here that at least half of the total SuSy of developing cotton studying synthesis of these 13-glucans (12). In searching for the fibers (Gossypium hirsutum) is tightly associated with the catalytic subunit of the cellulose or callose synthase, we plasma membrane. Therefore, this form of SuSy might serve demonstrated that the most abundant UDP-Glc binding to channel carbon directly from sucrose to cellulose and/or polypeptide in the cotton fiber plasma membrane was one of callose synthases in the plasma membrane. By using detached 84 kDa (13). Since this is close to the size of the catalytic and permeabilized cotton fibers, we show that carbon from subunit of the A. xylinum cellulose synthase (14), we purified sucrose can be converted at high rates to both cellulose and and partially characterized this polypeptide. We report that it callose. Synthesis of cellulose or callose is favored by addition has a molecular mass of 91 kDa (and will be referred to as p91) of EGTA or calcium and cellobiose, respectively. These find- and that p91 is a membrane-associated form of SuSy. Other ings contrast with the traditional observation that when data support a model in which this form of SuSy could exist in UDPglucose is used as substrate in vitro, callose is the major a complexwith ,B-glucan synthases and serve to channel carbon product synthesized. Immunolocalization studies show that from sucrose via UDP-Glc to cellulose and/or callose. SuSy can be localized at the fiber surface in patterns consis- tent with the deposition of cellulose or callose. Thus, these MATERIALS AND METHODS results support a model in which SuSy exists in a complex with the j8-glucan synthases and serves to channel carbon from Photolabeling and Purification of SuSy. Fibers of Gos- sucrose to glucan. sypium hirsutum Acala SJ-2 were harvested 21 days after anthesis; membrane and soluble proteins were separated and The well-characterized cellulose synthase from the bacterium photolabeled with [32P]UDP-Glc (13). Membrane-bound p91 was purified by solubilization of membrane proteins with 1% Acetobacter xylinum is a plasma-membrane-localized enzyme digitonin and fractionation by rate-zonal glycerol-gradient that clearly uses UDPglucose (UDP-Glc) both in vivo and in centrifugation (13). Fractions enriched in photolabeled p91 vitro as substrate for synthesis of 13-1,4-glucan microfibrils (1). were pooled and further fractionated by SDS/PAGE (ref. 15; The high levels (2) and turnover rate (3) of UDP-Glc also 1.5-mm thick gels; stacking and separating gels, 4.5% and 7.5% suggest that it is the substrate for higher plant cellulose polyacrylamide, respectively). For sequencing, gels were pre- synthesis. However, when isolated plasma membranes of pared the day before use, and the running buffer contained 0.1 higher plants are supplied with UDP-Glc, the major product mM thioglycollate. The region of the abundant, photolabeled synthesized is usually not cellulose but callose (j3-1,3-glucan; p91 was identified by radioautography and Coomassie blue ref. 4). A recent study with developing cotton fibers (5) has staining, and the corresponding region from other lanes was shown that a subfraction of membrane proteins can synthesize excised and solubilized in SDS sample buffer (yield of 1 ,ug of a higher ratio of cellulose to callose, but the rate of cellulose purified p91 per lane). About 40 ,ug of p91 was recovered for synthesis was far below that observed in vivo. microsequencing and 200 ,ug was recovered for polyclonal In plants, UDP-Glc can potentially be synthesized by two antibody production in rabbits. Purified p91 showed only a different pathways. One route involves the enzyme UDP-Glc single spot on two-dimensional gels (16). pyrophosphorylase (EC 2.7.7.9). Levels of this enzyme are Quantification of SuSy in Membranes and Soluble Frac- usually very high in plant cells, but it probably functions tions. Equal amounts of protein from soluble and membrane primarily in the direction of UDP-Glc degradation, particu- fractions were electrophoresed and p91 was quantified by larly in nonphotosynthetic tissues (6). The second route in- Western blot analysis as described (17), except with Amersham volves the enzyme sucrose synthase (SuSy; EC 2.4.1.13). Like ECL detection (primary anti-SuSy or preimmune serum, di- the phosphorylase reaction, the reaction catalyzed by SuSy is luted 1:3300; secondary goat anti-rabbit peroxidase, diluted freely reversible, but the high levels of this enzyme and 1:20,000) followed by scanning densitometry. The densitom- steady-state measurements of levels of its substrates and etry values and the relative amounts of total protein in products in nonphotosynthetic tissues suggest that it functions membrane and soluble fractions were then used to calculate primarily in the direction of sucrose degradation and UDP-Glc the percent p91 in membranes as a function of fiber age. synthesis (7, 8). SuSy has formerly been studied as a cytoplas- Activity for SuSy was measured in direction of sucrose cleavage mic enzyme that provides carbon for respiration and cell wall in 60-,ul reaction mixtures [40 mM Mes-KOH, pH 6.8/12.5 mM polysaccharide and starch synthesis (9, 10). Evidence for a sucrose/3 mM UDP/0.05% digitonin/1 mM dithiothreitol 5 ,ug biosynthetic role of SuSy is provided by substantially reduced Abbreviations: SuSy, sucrose synthase; FITC, fluorescein isothiocya- The publication costs of this article were defrayed in part by page charge nate. payment. This article must therefore be hereby marked "advertisement" in *To whom reprint requests should be addressed. accordance with 18 U.S.C. §1734 solely to indicate this fact. §Deceased July 4, 1995. 9353 Downloaded by guest on September 27, 2021 9354 Plant Biology: Alilor et al. Proc. Natl. Acad. Sci. USA 92 (1995) of either membrane or soluble proteins, dialyzed against 5 mM Mes-KOH (pH 7.5)]. Reactions were incubated 30 min at 30°C and fructose was quantified as described (18). Reactions lacking UDP were used to correct for any reducing sugar produced by invertase. Microsequencing. SDS sample buffer containing pure p91 was exchanged to 0.1 M NaHCO3/0.5% CHAPS by repeated concentration (Centricon-10 filter). p91 (25 ,ug/0.1 ml) was treated with 2 ,ug of trypsin (Boehringer Mannheim) at 37°C for 30 min, and the reaction was terminated with 1 ,lI of 0.01% trifluoroacetic acid. Tryptic peptides were separated by HPLC 3 4 5 6 and the two best-separated and most abundant peptides were FIG. 1. SDS/PAGE characterization of p91 from cotton fiber microsequenced by using an Applied Biosystems microse- membranes. Lanes: 1 and 3, Coomassie blue staining of proteins; 2 and quencer at Calgene, Inc., Davis, CA. 4, p91 photolabeled with [32P]UDP-Glc; 1 and 2, crude membrane Synthesis of j8-Glucans in Detached Cotton Fibers. Fibers proteins; 3 and 4, purified p91; 5 and 6, Western blot analysis of equal [-2 mg (dry weight)] from fresh 22- to 27-day bolls of amounts of crude soluble (lane 5) or membrane (lane 6) proteins with greenhouse-grown Acala SJ-2 cotton were excised and imme- p91 antibody. diately placed in 0.25-ml reaction mixtures [40 mM Mes-KOH, pH 6.8/0.01% digitonin/30 mM [U-14C]sucrose (0.5 ACi/ observed (Olympus BH-2 microscope) by using fluorescence mmol; 1 Ci = 37 GBq)] and other components as indicated. filter IB with a 15-nm band pass at 495 nm and a Zeiss KP560 After incubation (30°C, 10 min), reaction mixtures were acid- barrier filter. Images were photographed with Kodak T-Max ified to pH 2 with HCl, heated 5 min at 100°C, filtered onto 400 film. glass-fiber filters, and washed briefly with water and chloro- form/methanol, 1:2 (vol/vol). Fibers were dried, weighed, RESULTS homogenized in water (Omni TH tissue homogenizer), refil- tered, and washed extensively with water, and radioactivity was Identification of a Membrane-Associated Form of SuSy. measured by scintillation counting. When crude cotton fiber membrane preparations (Fig. 1, lane Analysis of Reaction Products. Product (3000 cpm, five 1) were photolabeled with [32P]UDP-Glc in the presence of replications) was incubated 4 days at 37°C in 50 mM sodium Mg2+, only a 91-kDa polypeptide (p91) was detected after acetate (pH 4.5) containing 5 mM NaN3 with (i) no enzyme short exposure of radioautograms (Fig. 1, lane 2). This (control), (ii) endo-1,4-,B-glucanase (purified to homogeneity polypeptide was purified to relative homogeneity as judged by from Trichoderma reesei; 2 units), or (iii) exo-1,3-f3-glucanase Coomassie blue staining (Fig.