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. 1, lane 3) and still possessed the (purified to homogeneity from Trichoderma SP; 1 unit). These photolabeled [32P]UDP-Glc (Fig. 1, lane 4). Antibody against glucanases (obtained from Megazyme, Sydney, Australia) purified p91 reacted specifically and with roughly equal inten- displayed the expected specificity to degrade A. xylinum cel- sity with a 91-kDa polypeptide in crude soluble or membrane lulose or pachyman (1,3-,3-glucan) (data not shown). Reaction proteins (Fig. 1, lanes 5 and 6). No reaction was observed with products were analyzed by filtering and measuring radioactiv- preimmune serum (data not shown). ity in the product remaining after digestion. The homogenized Microsequencing of two tryptic fragments of purified p91 and washed product (5000-10,000 cpm, two replications) was indicates that p91 is a form of SuSy, judging from the high dried and subjected to methylation analysis as described (19). sequence homology with known plant SuSy sequences (Fig. 2). Radioactive derivatives were separated (with similar results) Its molecular mass (23, 24), ability to bind UDP-Glc, cross- by GLC equipped with a stream splitter (20) or by TLC on reactivity with an antibody against purified maize SuSy (data silica gel G by using hexane/ethyl acetate, 1:1 (vol/vol), as not shown), and direct activity measurements also support the solvent. Derivatives from unlabeled cellulose, cellobiose, and presence of SuSy in the membrane fraction. Its specific activity pachyman were used as standards for terminal Glc and 3- or measured in the direction of sucrose cleavage (mean of five 4-linked Glc. These standards, as well as the predominant experiments) was 0.32 and 0.14 nmol of fructose formed per 4-Glc derived from endogenous fiber cellulose in reaction min per mg of fiber (dry weight) for the membrane and soluble mixtures, were detected by charring (46) thin-layer chromato- fractions, respectively. grams, after measuring radioactive derivatives by using a The tight association of SuSy with membranes is not yet well FUJIX BioImaging analyzer (Tokyo). understood. When antibody quantification was used, no SuSy Immunolocalization of SuSy. Ovules of G. hirsutum Acala was washed from membranes by using 0.5 M NaCl, the SJ-1 were cultured in vitro until days 18-21 of fiber develop- chaotrope 0.5 M KI, 1% deoxycholate, or 25 mM EDTA. ment at 34°C as described (21). Western blot analysis of About half was released by high pH (>10), 3 M urea, or 1% proteins from cultured fibers showed a specific reaction of Triton X-100. Only strong detergents (1% digitonin, CHAPS, anti-SuSy antibody, but not preimmune serum, with one or SDS) caused complete release. However, the enzyme does polypeptide as did proteins from plant-grown fibers (see not partition into Triton X-114, suggesting that it is not a below). For indirect immunofluorescence, fibers attached to transmembrane protein (25). At least half of SuSy, as quan- ovules were fixed for 1 hr at room temnperature in 3.7% tified either by enzyme activity or antibody reaction, was (vol/vol) formaldehyde/50 mM Pipes, pH 7.0/5 mM MgSO4/5 mM EGTA/0.5% Tween 20 (for Fig. 4 a-e) or in Histochoice Potato, res 706-713 S G F H I D P Y Trypsinfragment, cotton S G F N I D P Y (Amresco, Solon, OH), which was better for preserving de- Maize res S L H I D P Y tailed structure (for Fig. 4 f and g). This was followed by Sh, 706-713 G permeabilization as described (22). After 10 min in 0.1% Potato, res 381-396 FEVWPYMETFIEDVAK sodium borohydride, fibers were incubated overnight at 4°C in Trypsinfragment, cotton F EVWPYLETYTEDVAG preimmune or anti-SuSy serum (diluted 1:200 for Fig. 4 a-e Maize Sh, res 378-393 FDVWPYLBTYTEDVSS and 1:100 for Fig. 4 f and g). The second antibody was FIG. 2. Comparison of amino acid sequences of two tryptic pep- fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit tides of the cotton fiber membrane-associated p91 (SuSy) with ho- IgG (diluted 1:200, 1 hr at room temperature). Mounting mologous SuSy sequences derived from cDNA clones of potato medium was 0.1% phenylenediamine in 90% glycerol/10% (GenBank accession no. M18745) and Maize Sh (endosperm-specific) Tris-buffered saline (pH 8.6) (vol/vol). FITC fluorescence was SuSy (GenBank accession no. X02382). Downloaded by guest on September 27, 2021 Plant Biology: Amor et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9355 degrades these glucans also did not degrade the radioactive product (data not shown). Further characterization of the gc 75 ~~~~ _ -:~oker130 reaction products by methylation analysis and glucanase di- gestions (Table 1) showed that, in the absence of Ca2", 55-65% of the product is cellulose (f3-1,4-glucan) and the remainder is callose (,B-1,3-glucan). The only radioactive de- rivatives detected either by TLC or GLC were characteristic of 3- or 4-linked Glc. About 50-70% of the f3-1,4-glucan product 5- is insoluble in 24% (wt/vol) KOH or acetic nitric reagent (29); therefore, it has some crystallinity and can be called cellulose. However, when Ca2+ and cellobiose, two effectors required to 0 activate cotton fiber callose synthase (30), are present, syn- 9 16 20 24 28 thesis of callose predominates. Days after anthesis Immunolocalization of SuSy in Cotton Fibers. In situ im- munolocalization using anti-SuSy antibody was performed by FIG. 3. Percent of membrane-associated SuSy (p91) as a function using fibers on cotton ovules cultured in vitro, which facilitate of fiber development. Secondary wall synthesis begins between 15 and manipulations and allow rapid fixation with less damage. Such 17 days after anthesis in these fibers (27). fibers undergo development and secondary wall formation associated with the plasma membrane, partitioning into the similar to plant-grown fibers, although secondary wall synthe- occurs was seen upper PEG-rich phase during two-phase extractions in dex- sis earlier in culture (27). A positive reaction tran/PEG (26). in some, but not all, fibers (Fig. 4 a and b), which was not observed with the preimmune serum (Fig. 4 c and d). A A substantial percent of p91, as quantified by antibody punctate pattern of localization of SuSy was occasionally reaction, was found in the membrane fraction of Acala SJ-2 or Coker 130 varieties of G. hirsutum at all stages of fiber detected (Fig. 4e) in a pattern reminiscent of callose deposition in perturbed plant cells (31) including in vitro cotton fibers development (Fig. 3). In addition, p91 (as well as many other polypeptides characteristic of isolated plasma membranes, (E. M. Roberts and C.H.H., unpublished data). However, fluorescence was more frequently observed to be concentrated including callose synthase) can also be extracted with 2% in striations paralleling the secondary wall microfibrils (Fig. 4 (wt/vol) SDS from well-washed fiber walls, particularly during f and g). These striations traversed both sides of the fiber in a secondary wall synthesis. During this time, the amount of SuSy helix, and they occurred in never-dried fibers and those that in washed walls can that found in approach membranes (data were dried to the slide before antibody reaction. (Fig. 4 f and not was shown). p91 also demonstrable by Western blot g is from dried fibers so that more striations are in the same analysis of proteins from membranes of cotton roots, etiolated focal plane.) Fluorescence always occurred across the fiber pea and bean stems, and cultured tobacco cells (data not width, even after plasmolysis where differential interference shown). contrast microscopy revealed a shriveled protoplast (data not Synthesis of Cellulose and Callose from Sucrose in De- shown). This implies that a SuSy-synthase complex may re- tached and Permeabilized Cotton Fibers. To test whether this main wall-associated after plasmolysis, a possibility supported form of SuSy may channel carbon directly from sucrose to by our finding of substantial SuSy protein extractable from the glucan synthase complexes, cotton fibers were detached from wall fraction (see above). ovules, permeabilized with digitonin, and supplied with [14C]sucrose as substrate. Incorporation into polymers was observed at rates that were usually higher than those observed DISCUSSION for the synthesis of callose in vitro from UDP-Glc and some- The discovery of substantial SuSy associated with the plasma times approached rates of cellulose synthesis in vivo at this membrane of cotton fibers and other plant cells was surprising. stage of fiber development (Table 1). However, the coupled Although SuSy has always been studied as a soluble enzyme, reaction is labile, terminating within 10 min, rates are often there are a few clues from the literature suggesting a mem- variable, and the reaction is not reproducibly demonstrable brane-associated form. Helsper (32) reported that sucrose was with frozen-thawed fibers or in isolated membranes. There- synthesized from UDP-Glc by isolated membranes of Petunia fore, the rates given for freshly harvested fibers are shown as hybrida pollen tubes. A 92-kDa polypeptide on the cytoplasmic the mean and the range of activities from nine experiments. face of the plasma membrane, which might be SuSy, is among No reaction product was solubilized by 1% SDS or by those enriched with callose synthase from Beta vulgaris L. (33). a-amylase treatment. Dicotyledenous plants do not synthesize The nature of the strong association of SuSy with the plasma mixed-linked ,-1,3/1,4-glucans (28), and a lichenase that membrane is not yet clear. From studying the amino acid Table 1. Synthesis of ,3-glucans by digitonin-permeabilized cotton fibers Reaction rate, nmol Product analysis per min per mg (dry wt) Enzyme digestion Methylation analysis Reaction conditions Mean Range % 3-1,4-G % 03-1,3-G % 03-1,4-G % f3-1,3-G [14C]Sucrose/EGTA 0.28 0.06-0.78 55-65 35-45 55 45 [14C]Sucrose /CaCl2/CB 0.24 0.06-0.55 30-40 60-70 40 60 UDP-[14C]Glc/CaCl2/CB 0.09 0.03-0.10 10-15 85-90 <10 >90 UDP-[14C]Glc/EGTA 0.01 0.01-0.02 ND ND ND ND Cellulose synthesis in vivo 0.7 -1.5 Effectors were added at the following concentrations: EGTA, 25 mM; CaCl2, 1 mM; CB (cellobiose), 10 mM. For comparison with other previous studies (e.g., refs. 5 and 47), a rate of 1 nmol ofglucose per min per mg (dry wt) is approximately equivalent to 450 nmol of glucose per min per mg of membrane protein. ,3-1,4-G, 13-1,4-glucan (celluose); f3-1,3,-G, ,3-1,3-glucan (callose); ND, not determined. Cellulose synthesis in vivo was calculated for 22-27 days after anthesis from the data of Meinert and Delmer (27). Downloaded by guest on September 27, 2021 9356 Plant Biology: Amor et al. Proc. Natl. Acad. Sci. USA 92 (1995)

FIG. 4. In situ immunolocalization of SuSy in cotton fibers. (a, c, and f) Differential interference contrast images. (b, d, e, and g) FITC fluorescence images of anti-rabbit IgG-FITC that reacted with anti-SuSy (b, e, and g) or preimmune serum (d). (Bars: a for a-d, 50 ,um; in e and in f for f and g, 10 ,um.) Times of micrographic exposure and negative printing were the same for b and d. sequence derived from a recently isolated cotton SuSy cDNA of Ca2+ and a ,B-glucoside such as cellobiose favors callose (L. Perez-Grau and D.P.D., unpublished data) and from other synthesis from sucrose, whereas chelation of Ca2+ from the plants, we have not found sequences indicative of prenylation system favors cellulose synthesis. These results correlate log- or myristylation sites, common modifications that often de- ically with the situation in vivo, where an increase in intracel- termine membrane localization (34, 35). However, SuSy can be lular Ca2+ is believed to inhibit cellulose synthesis and activate phosphorylated (36). callose synthase (41). In contrast, Kudlicka et al. (5) proposed The existence of high levels of membrane-associated SuSy in that Ca2+ and cellobiose (as well as Mg2+ and cGMP) en- cotton fibers engaged in high-rate cellulose synthesis in vivo hanced synthesis of both polymers from UDP-Glc in vitro. suggested that this enzyme might associate with cellulose Although synthesis of both cellulose and callose from su- synthase and channel carbon directly from sucrose (via UDP- crose has clearly been demonstrated, the system is not opti- Glc) to growing glucan chains. Metabolic channeling has many mized. For the work reported herein, the most important advantages (37). (i) In this case, channeling of UDP-Glc finding is that there was very little variability among replicates directly to cellulose synthase would ensure high availability of in the linkages formed and in the effect of EGTA or Ca2+ and a substrate used in many other cellular reactions. (ii) The UDP cellobiose in varying the ratio of cellulose to callose produced. produced by the synthase could be directly recycled by SuSy The extreme lability of the system and variable reaction rates and never accumulate to high levels; UDP has been shown to suggest that a higher level of organization may be required, inhibit the A. xylinum cellulose synthase (1) and higher plant that critical enzymes are subject to degradation, and/or that callose synthase (38). (iii) The unusually high free energy of critical effectors are limiting in disrupted plant cells. The hydrolysis of the sucrose linkage would be conserved and used requirement for higher levels of organization is supported by for glucan polymerization. In contrast, synthesis of UDP-Glc the fact that high percentages ofmembrane-bound SuSy can be via UDP-Glc pyrophosphorylase requires two ATP equiva- extracted from well-washed walls. Therefore, the wall and the lents. Energy conservation could be especially important for plasma membrane may be closely coupled when membrane- cotton fibers in which "80% of the carbon supplied is con- bound SuSy is active, and such a relationship could be partially verted to cellulose during secondary wall synthesis (39). disrupted by fiber detachment and permeabilization. The demonstration that permeabilized cotton fibers can The lability and variability in reaction rates have prevented synthesize 3-glucans from sucrose supports this model. To the us from presenting firm quantitative data concerning optimal best of our knowledge, this is the first case in which substantial reaction conditions. However, we have never observed sub- levels of cellulose have been synthesized at high rates in stantial competition for incorporation of radioactivity from disrupted plant cells. The reason for this success seems to lie sucrose into glucan by addition of unlabeled UDP-Glc, sug- in the use of sucrose, rather than UDP-Glc, as substrate, since gesting this intermediate may be channeled directly to the UDP-Glc generally favors callose synthesis in vitro (4). Such synthase complexes. We also have never observed either results imply that the cellulose synthase preferentially accepts stimulation or inhibition of the reaction by addition of UDP, UDP-Glc channeled via sucrose degradation, whereas the the second substrate of SuSy, suggesting that sufficient UDP callose synthase can accept it either in its free form or from is present and can be recycled in the coupled reaction as is SuSy. Thus SuSy might associate with the two synthases characteristic of intermediates involved in metabolic channel- independently or with a single complex capable of synthesis of ing (37). Alternatively, any stimulation of SuSy by added UDP either cellulose or callose, as suggested by Jacobs and North- could have been canceled by a concomitant inhibition of cote (40) and Delmer and Amor (41). Furthermore, addition glucan synthase. Furthermore, magnesium pyrophosphate, Downloaded by guest on September 27, 2021 Plant Biology: Amor et al. Proc. Natl. Acad. Sci. USA 92 (1995) 9357 which inhibits flow of carbon to UDP-Glc via UDP-Glc 4. Delmer, D. P., Ohana, P., Gonen, L. & Benziman, M. (1993) pyrophosphorylase and stimulates sucrose degradation via Plant Physiol. 103, 307-308. SuSy (42), always showed only stimulation of the coupled 5. Kudlicka, K., Brown, R. M., Jr., Li, L., Lee, J. H., Shin, H. & Kuga, S. (1995) Plant Physiol. 107, 111-123. reaction (unpublished data). However, these findings must 6. Kleczkowski, L. (1994) Phytochemistry 37, 1507-1515. remain tentative until the reaction is further stabilized and the 7. Xu, D.-P., Sung, S.-J., Loboda, T., Kormanik, P. P. & Black, C. C. variable reaction rates are minimized. (1989) Plant Physiol. 90, 635-642. The immunolocalization patterns of SuSy are consistent 8. Geigenberger, P., Langerberger, S., Wilke, I., Heineke, D., Heldt, with roles in callose synthesis (punctate pattern) and cellulose H. & Stitt, M. (1993) Planta 190, 446-453. synthesis (striated pattern). The punctuate pattern was ob- 9. Tobias, R. B., Boyer, C. D. & Shannon, J. C. (1992) PlantPhysiol. served rarely, suggesting that it might be associated with cell 99, 146-152. perturbation and callose synthesis before fixation. The stria- 10. Zrenner, R., Salanoubat, M., Willmitzer, L. & Sonnewald, U. (1995) Plant J. 7, 97-107. tions were observed more frequently and always paralleled the 11. Chourey, P., Chen, Y. C. & Miller, M. E. (1994) Maydica 36, directions of helical secondary wall microfibril deposition and 141-146. even shifted direction at the "reversals" in microfibril helix 12. Maltby, D., Carpita, N. C., Montezinos, D., Kulow, C. & Delmer, direction that are characteristic of cotton fibers (data not D. P. (1979) Plant Physiol. 63, 1158-1164. shown). The relatively wide striations in our differential in- 13. Delmer, D. P., Solomon, M. & Read, S. M. (1991) Plant Physiol. terference contrast and fluorescent images (0.18-0.36 ,um) 95, 556-563. could correlate with similarly sized and oriented microfibril 14. Lin, F.-C., Brown, R. M., Jr., Drake, R. R., Jr., & Haley, B. E. (1990) J. Biol. Chem. 265, 4782-4784. bundles observed in fiber secondary walls (43). Synthesis of 15. Laemmli, U. K (1970) Nature (London) 227, 680-685. microfibril bundles would require enrichment of the relevant 16. Andrawis, A., Solomon, M. & Delmer, D. P. (1993) Plant J. 3, enzymes along the bundle, which could explain the striated 763-772. alignment of SuSy. In this configuration, SuSy might not always 17. Elthon, T. E. & McIntosh, L. (1987) Proc. Natl. Acad. Sci. USA appear punctate after indirect immunofluorescent imaging 84, 8399-8403. because of the high density along the striations. As an analogy, 18. Lever, M. (1972) Anal. Biochem. 47, 273-279. in differentiating tracheary elements that synthesize cellulose- 19. Blakeney, A. B. & Stone, B. A. (1985) Carbohydr. Res. 140, rich secondary wall thickenings, freeze-fracture electron mi- 319-325. 20. Ohana, P., Delmer, D. P., Volman, G., Steffens, J. C., Matthews, croscopy has revealed plasma membrane particle rosettes at D. E. & Benziman, M. (1992) Plant Physiol. 98, 708-715. densities up to 191 rosettes per ,um2 at the thickening sites (44). 21. Haigler, C. H., Rao, N. R., Roberts, E. M., Huang, J.-Y., Up- Further support for the association of SuSy with sites of callose church, D. P. & Trolinder, N. L. (1991) Plant Physiol. 95, 88-96. and cellulose synthesis comes from preliminary immunolocal- 22. Uhnak, K. S. & Roberts, A. W. (1995) Protoplasma, in press. ization studies in differentiating tracheary elements of Zinnia 23. Su, J. C. & Preiss, J. (1978) Plant Physiol. 61, 389-393. that show that SuSy is localized at the cell plate (a site ofcallose 24. Delmer, D. P. (1972) J. BioL Chem. 247, 3822-3828. deposition; ref. 45) in dividing cells and under bands of intense 25. Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607. 26. Larsson, C., Widell, S. & Kjellbom, P. (1987) Methods Enzymol. cellulose deposition at the stage of secondary wall deposition 148, 558-568. (C.H.H., S.J., Y.A., and D.P.D., unpublished data). 27. Meinert, M. & Delmer, D. P. (1977)PlantPhysiol. 59,1088-1097. In sum, the discovery of this membrane-bound form of SuSy 28. Carpita, N. C. & Gibeaut, D. (1993) Plant J. 3, 1-30. and the ability to synthesize cellulose from sucrose at some- 29. Updegraff, D. M. (1969) Anal. Biochem. 32, 420-424. times substantial rates in disrupted plant cells offer additional 30. Hayashi, T., Read, S. M., Bussell, J., Thelen, M. R., Lin, F.-C., approaches for the characterization of the pathway for cellu- Brown, R. M., Jr., & Delmer, D. P. (1987) Plant Physiol. 83, lose synthesis in plants. 1054-1062. 31. Dietrich, R. A., Delaney, T. P., Uknes, S. J., Ward, E. R., Ryals, J. A. & Dangl, J. (1994) Cell 77, 565-577. This was the last manuscript communicated by Dr. Joseph E. Varner 32. Helsper, J. P. F. G. (1979) Planta 144, 443-450. (deceased July 4, 1995), and thus we wish to publish this manuscript 33. Wu, A. & Wasserman, B. P. (1993) Plant J. 4, 683-695. in his honor to recognize his long and distinguished career in plant 34. Mcllhinney, R. A. J. (1990) Trends Biol. Sci. 15, 387-390. biology and his exemplary collegiality. We thank J. Bleibaum (Cal- 35. Clarke, S. (1992) Annu. Rev. Biochem. 61, 355-386. gene, Inc., Davis, CA) for separation and microsequencing of tryptic 36. Shaw, J. R., Ferl, R. J., Baier, J., St. Clair, D., Carson, C., peptides. Polyclonal antibody against maize Sus2 SuSy was the gen- McCarty, D. R. & Hannah, L. C. (1994) Plant Physiol. 106, erous gift of P. Chourey, University of Florida. The biochemical 1659-1665. studies were supported by a contract to D.P.D. from the U.S. Depart- 37. Ovadi, J. (1991) J. Theoret. Bio. 152, 1-22. ment of Energy and a grant from The United States-Israel Binational 38. Morrow, D. & Lucas, W. J. (1986) Plant Physiol. 81, 171-176. Agricultural Research and Development Fund (BARD). The immu- 39. Mutsaers, H. J. (1976) Ann. Bot. 40, 300-315. nolocalization studies were supported by grants to Texas Tech Uni- 40. Jacobs, S. R. & Northcote, D. H. (1985) J. Cell Science Suppl. 2, versity (TTU) from the Howard Hughes Medical Institute/ 1-11. Undergraduate Biological Sciences Education Program and National 41. Delmer, D. P. & Amor, Y. (1995) Plant Cell 7, 987-1000. Science Foundation Research Experiences for Undergraduates Pro- 42. Delmer, D. P. (1972) Plant Physiol. 50, 469-472. gram (for S.J. and M.W.) and by grants to C.H.H. from the Texas 43. Willison, J. H. M. & Brown, R. M., Jr. (1977) Protoplasma 92, Advanced Research program, Cotton Incorporated, and the TTU 21-41. Institute for Plant Stress Research. 44. Herth, W. (1985) Planta 164, 12-21. 45. Stone, B. 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