cw '~ :IKEI'. “*1 ‘1‘.” ‘+ I; ‘ ":l' I I I I" I‘m. "In I ' I I 'I IIIEIs. . I'I ' V ' . U. \ I.” II I I ' I ".I0'O\'v‘d'.l .IIII'I” ‘ " I-."' ".‘,’ :I-I . , r' VIII ”“1“.“ ‘3‘ :I .\ . I‘- I had.” . H‘. .." ' 1 | I if: .: IV N'II ' - WINT- I ‘IUIcilI.¢“$ W I” I: ' '- ' I‘LL 1 .I\ "W. P: I: U V ‘ TL'O.‘ . 3' .|’ I . .3 \d;:__ r.m\_”~,:' 3 ,£3. bag 7 . $5453?- «7;, II 'I ‘ I" _ - _ I. . a 1-3? . $0 ‘:~ ‘ ",:,r #5..- . I‘ JVM I ' -. :II'341J'M’: ' I I I‘ a" . 7 7 3 ‘ | f “' M. 5:. . I 3' . V A ”I. '. - . 7 I . I}. .SII'IEY'W aw I" A." | If g)? I :‘I “:3. I' '1‘..(‘III.‘.IV I ' . ‘ I . l I - Him. II II 3::5. M. "I.“ l I I I :IJI " II‘ IJII' "$le .III- ~ "ILZI. "M" 'IJ‘II .I‘ o IIII 17221335 This is to certify that the thesis entitled GLUEAN SYNTHESIS IN SOYBEAN CELLS: PROPERTIES OF THE ENZYMES INVOLVED IN DEPOSITION OF GLUCANS DURING CELL WALL REGENERATION presented by Anita Sherrie Klein has been accepted towards fulfillment of the requirements for P-h. D. degree in Biochemistry Major professor 0-7639 " “new . ‘2 . “‘f”’,i OVERDUE FINES: 25¢ per day per item . RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation record GLUCAN SYNTHESIS IN SOYBEAN CELLS: PROPERTIES OF THE ENZYMES INVOLVED IN DEPOSITION OF GLUCANS DURING CELL WALL REGENERATION By Anita Sherrie Klein A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1981 5"?8 €//é ABSTRACT GLUCAN SYNTHESIS IN SOYBEAN CELLS: PROPERTIES OF THE ENZYMES INVOLVED IN DEPOSITION OF GLUCANS DURING CELL WALL REGENERATION. By Anita Sherrie Klein The early events in cell wall regeneration have been examined, using protoplasts derived from liquid suspension cultures of Glycine max. Without a detectable lag, supplied 14C-glucose is incorporated into acetic and nitric acid-resistant material. This material has been identified chemically as cellulose by several independent techniques. Cytological observations indicate that there are no cellulose microfibrils present on the surface of soybean protOplasts, fixed in glutaraldehyde immediately after isolation. However after incubating protoplasts for 1 h in cell wall regeneration medium, numerous microfibrils are visible on the outer surface of the plasma membrane. These indicate that the supplied 14C-glucose is incorporated into new cellulose microfibrils, not pre-existing fibrils. During the first three h of wall regeneration, protoplasts also synthesize a wide range of polysaccharides and polysaccharide-containing polymers which qualitatively resemble the pectic and hemicellulosic polymers found in the cell walls and recovered from the Spent culture medium of cultured soybean cells. While no appreciable quantities of 3-B-glucan are found in the cell walls or extracellular polysacharides of cultured soybean cells, protoplasts and intact cells utilize exogenous UDP-[14CJ-glucose to synthesize 3-8-glucan. An attempt was made to isolate from soybean protoplasts a preparation of plasma membrane vesicles in which the topology was unaltered (i.e. periplasmic face remained facing outward. These vesicles were isolated by affinity chromatography on concanavalin A Sepharose. These vesicles utilize supplied UDP-glucose to synthesize 3-B-glucan, suggesting that the 3-B-glucan synthetase can accept substrate from the periplasmic surface of the cell. In addition, the role of the plasma membrane in modulation of enzyme activity is discussed. Results are described wherein the activity of the 3-e-glucan synthetase in membrane vesicles, derived from protoplasts, is stimulated by the induction of a transmembrane potential across the vesicles. Under conditions where protoplasts were competent to synthesize cellulose from glucose, UDP-glucose supplied to the periplasmic surface of the plasma membrane did not serve as an effective substrate of the cellulose synthetase. These results are discussed in relation to various models for the orientation of glucan synthetases in the plasma membrane. As a first step in the purification and characterization of the glucan synthetases of soybean cells, procedures were devel0ped to prepare soluble forms of the enzymes. Over 75% of the glucan synthetases of a crude membrane preparation are solubilized by a 15 min. treatment with 30 mM cholate at pH 7.8. Enzyme activity is stimulated and stabilized by high concentrations of glycerol. The glucan synthesized by the soluble enzyme preparation were analyzed by methylation analyses; the results indicate that at least two UDP-glucose: glucan synthetases, producing 3-linked and 4-linked glucosyl residues are present in the soluble enzyme preparation. ACKNOWLEDGEMENTS I would like to express my graditude to Dr. Deborah P. Delmer for her time and effort expended on my behalf during my sojourn in the land of carbohydrate chemistry. I am most thankful for advise and moral support to: Dr. Peter S. Carlson, who with Dr. Delmer served as my joint thesis advisors; Dr. Andrew Hanson and my other comnittee members Drs. Arnold Revzin and Allan Morris. My colleagues have taught me alot more than how to run a particular assay. I am particularly grateful to: Dr. Tony Bacic, who was a good friend, editor and motivating force while I wrote this thesis. I would also like to thank Carl Kulow, Kathy Johnson and Barbara Mitchell for their help during many stages of my research. In addition I would like to thank: Dr. David Montezinos, for guiding me through some crucial cytological experiments; Dr. Larry Fowke for providing me with detailed instructions for preparing samples for electron microscopy, Dr. Gerhard Weber for sending me the soybean culture SB-l, Dr. Shelagh Ferguson-Miller for advise on membrane protein purification and Charles Caldwell for sharing his expertise on membrane proteins and affinity chromatography. There is not enough room here for me to thank adequately, all of my friends who helped me attain this goal: my friends and colleagues at the Plant Research Laboratory and of 306 PBL, Dr. Stan Hattman, who provided inspiration and encouragement along with my many friends from ii the University of Rochester; my other Lansing friends, and my family, who let me pursue my dreams. All of you made this volume possible. This research was supported under Contract No. DE-AC02-7GER01338 for the U.S. Department of Energy. These funds are gratefully acknowledged. TABLE OF CONTENTS List of Tables List of Figures List of Abbreviations Introduction A. Cell Wall Composition 8. Cell Wall Regeneration 1. Chemical evidence for the synthesis of cellulose fibrils 2. Kinetics of cellulose synthesis by cultured protoplasts 3. Other cell wall constituents C. Cell Wall Biosynthesis D. Regulation of Biosynthesis Materials and Methods Chemicals and Reagents Cell Cultures ProtOplast Isolation Measurement of Water Potential iv ix xi L0 13 16 20 20 22 24 Materials and Methods cont. Isolation of Newly Synthesized Polymers 25 Cellulose Content 25 Determination of the Radioactivity in Crystalline Cellulose 25 Neutral Sugar Analyses 25 Methylation Analyses 27 Cell Counts 39 Protein Determinations 29 1. Protein content of soybean protoplasts 3o 2. Samples in cholate buffers 30 Preparation of Samples for Electron Microscopy 30 Preparation of Membrane-Bound Glucan Synthetases from 31 Soybean Cells Solubilization of the Glucan Synthetases 32 Glucan Synthetase Assay 32 Separation of Membrane Vesicles on ConA-Sepharose 33 1. Preparation of Concanavalin A Sepharose 6B 33 2. Preparation of membrane vesicles and affinity 34 chromatography Induction of Transmembrane Potentials Across Vesicles 35 Derived from Soybean Protoplasts. Estimated Counting Efficiency of Radioactive Samples 36 Chapter One: Early Events in Cell Wall Regeneration 37 A. Incorporation of 14C-glucose 37 Chapter One Cont. B. Cellulose Synthesis 1. Cellulose content of the cell wall of cultured soybean cells. 2. Chemical evidence that cellulose is synthesized during the first hours of wall regeneration. 3. Inhibition of cellulose synthesis by 2,6 dichlorobenzonitrile 4. The time course of cellulose synthesis 5. Deposition of fibrils on the surface of the plasma membrane C. Synthesis of Other Cell Wall Components 1. Neutral sugars 2. Methylation analysis 3. Synthesis of 3-e-glucan Discussion The Early Events in Cell Wall Regeneration Cellulose Synthesis Other Cell Wall Components Chapter Two: Solubilization of e-Glucan Synthetases Results A. Influence of pH and Sorbitol Concentration on Glucan Synthetase Activity in Crude Membrane Preparations B. Detergent Solubilization of Glucan Synthetases vi 38 38 41 46 46 51 6O 6O 64 67 68 68 68 69 72 72 72 75 Preparation of Solubilized Glucan Synthetases 1. General protocol 2. Product analysis D. Preliminary Experiments to Isolate Individual Glucan Synthetases. 1. Affinity chromatography on Reactive Blue Agarose 2. Gel filtration chromatography E. Additional Properties of the Solubilized Enzyme Preparation Discussion Chapter Three: Properties of the 3-B-Glucan Synthetase i_ situ Results and Discussion A. B. Glucan Synthesis from Exogeneous UDP-glucose Orientation of the 3-e-Glucan Synthetase in the plasma Membrane 1. Isolation of putative right-side-out plasma membrane vesicles by affinity chromatography 2. Isolation of putative inside-out plasma membrane vesicles The Influence of Membrane Potentials on Glucan Synthesis 1. Influence of potential on glucan synthesis in membrane vesicles 81 81 88 91 91 92 93 96 99 99 99 103 108 114 115 115 2. Influence of treatments designed to alter membrane potential on 3-5-glucan synthesis, from exogenous UDP-glucose, i_ viv O Discussion Orientation of the 3-B-Glucan Synthetase Influence of Membrane Potential on 3-3-Glucan Synthetase Activity Concluding Remarks Footnote References viii 121 123 123 125 127 129 130 1-1 1-2 1-3 2-1 2-2 3-1 3-2 3-3 3-4 3-5 LIST OF TABLES The effect of sorbitol and PEG 4000 concentration on the incorporation of glucose into cellulose and other ethanol-precipitable polymers by soybean protoplasts. The effect of DCB on the incorporation glucose. Neutral Sugar Composition A. Of cell walls and secreted polymers from soybean cell cultures (mole %). B. Distribution of radioactivity in the neutral sugar residues of newly synthesized polymers from protoplasts. Preparation of soluble glucan synthetases. Products of glucan synthetases. Utilization of exogenous UDP-glucose by whole cells and protoplasts. Stimulation of glucan synthesis from exogenous UDP-glucose by controlled lysis of protoplasts. Glucan synthetase activity of vesicles separated by conA sepharose chromatography Effect of treatments designed to induce transmembrane potentials on a-glucan synthesis in vesicle derived from soybean membranes. Influence of treatments designed to alter membrane potential on glucan synthesis in vivo. ix an 50 63 85 90 105 107 112 118 120 Chapter 1 1-1 1-2 1-3 1-4 1-5 1-6 1-7 1-8 Chapter 2 2-1 2-2 2-3 2-4 2-5 Chapter 3 3-1 3-2 LIST OF FIGURES Monosaccharide content of AN-resistant material. Saccharides released from AN-resistant material by cellulase digestion. Methylation analysis of AN-resistant material. Incorporation of 14C-glucose into cellulose and ethanol-precipitable polymerszshort term. Incorporation of 14C-glucose into cellulose and other polymers: 12 h. time course. Incorporation of 14C-glucose into cellulose: 24 h. time course. Time dependent deposition of fibrils on the surface of protoplasts. Methylation analyses of soybean polysaccharides. Effect of pH and sorbitol concentration on glucan synthetase activity from a crude membrane fraction. Influence of detergents on glucan synthetase activity and solubilization in membrane preparations. Effects of salt concentration on cholate-mediated solubilization. General protocol for preparing solubilized glucan synthetases. ’Time course of product formation by a solubilized preparation of glucan synthetases. Identification of glucan synthesized from exogenous UOP-glucose. Separation of membrane vesicles by affinity chromatography on conA Sepharose. X 43 45 48 53 55 57 59 66 74 77 83 95 AN reagent AN resistant AN soluble BTP conA DCB DTT ECM GC/MS GLC GDP-glucose HEPES MES nig NP-4O PEG POPOP PPO PMSF LIST OF ABBREVIATIONS acetic acid nitric acid reagent not hydrolyzed by acetic and nitric acid. solubilized by AN reagent 1,3-bis [tris (hydroxymethyl-methylamino-propane)] concanavalin A 2,6 dichlorobenzonitrile dithiothreitol extracellular material: ethanol precipitable- polymers from protoplast culture medium combined gas liquid chromatography and mass spectrometry gas liquid chromatography guanosine diphosphate glucose N-2-hydroxyethylpiperazine-N-Z-ethane sulfonic acid ZEN-morpholinOJethane sulfonic acid nigericin noniondet P-40 polyethylene glycol p-bis [2-(5-phenyloxazolylJ-benzene 2,5 diphenyloxazole paramethylsulfinyl fluoride xi TES N-tris [hydroxymethyl] methyl-Z-aminoethane sulfonic acid UDP-glucose uridine diphosphate glucose Y water potential val valinomycin xii Introduction Cell walls define the plant's shape, and capacity for growth, and act as a barrier to its external environment (Albersheim, 1976). Despite its importance as a major plant organelle, our understanding of the cell wall at a biochemical level is relatively limited (Darvill 33 31., 1980). Increasingly sophisticated analytical techniques are gradually revealing the complexity and dynamic nature of the covalent structure of polysaccharides and other macromolecular components of the cell wall (Darvill gt 1., 1980). Although our understanding of the chemistry of cell wall components is poor, even less is known about how these polymers are synthesized and subsequently secreted and assembled in the wall. Cell wall composition changes during different stages of development (Aspinall, 1973; Meinert and Delmer, 1977); it can also change in response to environmental stress (Boffey and Northcote, I975; McNairn, 1972; Smith and McCully, 1977). Changes in cell wall composition could reflect changes in substrate availability, modulation of enzyme activity or altered transcription/translation of polysaccharide synthesizing enzymes. Although many theories concerning the dynamic nature of the plant cell wall have been formulated an overall understanding of the underlying processes involved in wall assembly is lacking (Delmer, 1977). The composition of plant cell walls will be considered briefly as a framework for a discussion of cell wall regeneration and cell wall 1 2 biosynthesis. Cell wall regeneration provides advantages as a model system to elucidate cell wall structure and assembly: The individual components of the regenerating wall tend to be loosely associated and provide a more convenient material for some cytological and synthetic studies than the native cell wall (Willison 1976). The biosynthesis of cell wall polysaccharides will be reviewed, emphasizing particularly the problems involved in studying the synthesis of these polymers and the regulation of those polysaccharide-synthesizing enzymes which are associated with the plasma membrane . A. Cell Wall Composition The plant cell wall may be convienently described as a cellulosic lattice work bound together with an amorphous matrix of pectic and. hemicellulosic polysaccharides (Rees, 1972). Cell wall composition has been extensively reviewed by Albersheim (1976); Aspinall (1973); Beck and Wieczorek (1977); Darvill t al. (1980); Delmer (1977); and Wilkie (1979). Protein (especially glchprotein) is part of this cell wall matrix material and accounts for generally less that 10% of the cell wall by weight (Albersheim, 1976). Lignin, a complex polymer of cinnamyl alcohols, contributes rigidity to the wall and is generally found in the secondary cell wall, i.e. the cell wall of nondividing, fully differentiated cells (Albersheim, 1976). Native cellulose consists of linear 4-s-glucan chains which form extensive intra- and inter-molecular hydrogen bonds resulting in a crystalline fibrillar structure. Estimates of the degree of polymerization of the glucan-chains range from 2000 to more than 10,000 3 (Delmer, 1977). Cellulose usually represents from 10 to 50% of the dry weight of the cell wall (Albersheim, 1976; Delmer, 1977). The pectic and hemicellulosic polysaccharides of monocots differ significantly from those found in dicots. The primary cell wall of monocots is composed of less than 9% by weight pectic polysaccharides (Darvill gt al., 1980). These include arabinogalactan and a galacturonosyl- containing polymer. The pectic polysaccharides of dicots have been studied more extensively; they represent up to 30% of the weight of the cell wall. The major classes of dicot pectic polysaccharides are: rhamnogalacturonans, arabinogalactans, arabanans, galactans, and homogalacturonans. Little is known about the secondary, tertiary and quaternary structure of these polymers. Hemicellulosic polysaccharides of dicots account for 20 to 30% of the cell wall. The principal hemicellulosic polysaccharides are xyloglucans, a linear backbone of 4-B-glucan with xylosyl substituents at the C-6 position. Some of the xylosyl residues are linked to other heterosaccharides. Glucomannans are primarily chains of 4-e-glucosyl and mannosyl residues. The ratio of mannose to glucose (ca. 3:1) may vary between species and the degree of polymerization ranges from 100 to 400 (Beck and Wieczorek, 1977). They are generally considered secondary wall components, but may be present in small amounts in the primary cell wall of some dicots and monocots (Beck and Wieczorek, 1977). Hemicelluloses account for a greater proportion of the cell wall in monocots and are comprised of two major classes of polysaccharides: heteroxylans and mixed linkage B-glucans. Heteroxylans have a backbone of 4-8-linked D-xylopyranosyl residues substituted mainly with arabinofuranosyl residues. Mixed linkage 3,4-a-glucans are widely distributed in the cell walls of monocots. These are linear molecules; the distribution of 4 and 3-linked residues varies between individual polymers (ratio 4- to 3-linked: 2 to 5 fold; Wilkie, 1979). There have been two reports of mixed-linkage s-glucans in dicots: a water soluble glucan isolated from three day old hypocotyls of Phaseolus (Bachala and Franz, 1974), and the product of an ifl.!i££2 synthesizing system from a cell free extract of Pisum sativum (Brett and Northcote, 1975). However, these polysaccharides have not been isolated from other dicots, and are not generally considered to be constituent of the dicot cell wall (Darvill 35 al., 1980). In addition to the types of polysaccharides described above, 3-a-glucans (callose) are found in the cell walls of some tissues, both in dicots and monocots. 3-6-glucan has been found in the cell walls of pollen, pollen tubes and developing cotton fibers (Darvill gt 21-: 1980; Helep-Harrison, 1968). It is also found surrounding plasmodesmata and is rapidly synthesized in response to wounding and stress (Brett, 1978; Hughes and Gunning, 1980; McNarin, 1972; Smith and McCully, 1977). The degree of polymerization of these glucans has not been determined. No fibrillar structure or other gross organization has been attributed to the 3-3-glucan in 1119 (Heslop-Harrison, 1968); however, it is worthwhile to note that a particulate glucan synthetase preparation derived from Phytqphthora produced fibrillar 3-e-glucan in vitro (Wang and Bartnicki-Garcia, 1977). Liquid suspension grown cells have provided a convenient source of fairly uniform material for studies on the composition of the primary cell wall (Albersheim, 1976; Darvill gt al., 1980). In addition, such 5 cells in liquid culture, secrete polysaccharides and glycoproteins into the culture medium; some of these materials are similar to cell wall polymers (Albersheim, 1976). B. Cell Wall Regeneration Analysis of cell wall biogenesis may be facilitated by an examination of the process of cell wall regeneration by protoplasts (Willison 1976; Willison and Klein, 1981). Polysaccharide-degrading enzymes are used to strip the cell wall away from the plant protoplast; under the appropriate cultural conditions they begin to synthesize a new cell wall. Here the processes of biosynthesis and secretion are separable, to some extent, from cell wall assembly. One framework in which to consider cell wall regeneration is by comparison with the composition and structure of the native cell wall. Has the composition of cell wall polymers been altered? Are the individual constituents present in the same proportions in the regenerating wall and in the native cell wall? Is there coordination in the synthesis of individual wall polymers during regeneration, or does the process of removing the cell wall perturb the synthesis of some wall components but not others? How do the various polysaccharides and proteins interact to form a cell wall? Thus far there are no definitive answers to these questions. Nevertheless, characterization of the process of wall regeneration should provide useful insights about the organization and function of the cell walls of higher plants because it furnishes a mechanism to elucidate cell wall synthesis and assembly. 1. Chemical evidence for the synthesis of cellulose fibrils. The deposition of fibril-like material on the surface of isolated prot0plasts is one of the characteristics of cell wall regeneration and has been an important experimental system in cytological studies of cellulose synthesis. However, caution should be exercised before equating visualization of fibrils with the presence of newly-synthesized cellulose, i.e. crystalline 4-3-glucan. (Meyer and Herth, 1977; Willison 1976). Unbranched polysaccharides, particularly homopolymers, form fibril-like structures jn_yitgg (e.g. 4-3-mannan, 4-e-xylan of angiosperms (Preston, 1979; 3-8-glucan of fungi, Wang and Bartnicki-Garcia, 1976) Also, some of the noncellulosic components of the cell wall may exist as fibrils in situ (Preston, 1979). In many systems the regenerating wall will fluoresce when treated with Calcofluor White ST; however Calcofluor binding is not specific for 4-5-glucans and Calcofluor will stain the cell wall of a number of microorganisms which do not contain cellulose in their walls (Darken, 1961). Other criteria used as evidence that the regenerating wall contains crystalline cellulose are not unequivocal: Cellulase digestibility is not a rigorous test for the presence of cellulose because most available cellulase preparations contain other polysaccharide-degrading enzymes (Anderson and Ray, 1978). Similarly although alkali insolubility was the basis of the classical method for isolating 'pure' ascellulose, it is now well established that other cell wall polysaccharides are insoluble in hot alkali (Delmer, 1977). Nevertheless, in previous reports on the chemical composition of the regenerating cell wall, (Asamizu gt al., 1977; Asamizu and Nishi, 1980; Hanke and Northcote, 1974), one or the other of these procedures 7 was used as the sole means to demonstrate the presence of cellulose in the regenerating cell wall. In another study Vinca rosea protoplasts were cultured for two days. The regenerating wall was fully soluble in 24% KOH, indicating that it did not contain cellulose (Takeuchi and Komamine, 1978). Potentially, chemical inhibitors of cellulose synthesis could be used to verify the relationship between cellulose synthesis and fibril deposition. Coumarin (IOOuM) and 2,6-dichlorobenzonitrile (DCB) (IOuM) specifically inhibit cellulose synthesis in cultured cotton ovules. (Montezinos and Delmer, 1980). At higher concentrations these compounds also inhibit the synthesis of other cell wall polysaccha- rides. In an ultrastructural study, coumarin (0.5-1.5mM) inhibited fibril deposition on the surface of tobacco mesophyll protoplasts although after four days an aberrant wall was formed (Burgess and Linstead, 1977). Meyer and Herth, (1978) reported that coumarin (lmM) and DCB (5uM) totally inhibited cell wall formation by tobacco mesophyll protoplasts. Thus while these observations indicate that DCB or coumarin can certainly inhibit cellulose synthesis or fibril deposition, these compounds also seem to affect either the synthesis or assembly of other cell wall components, and the observations, described above, alone do not provide conclusive evidence that the fibrils are composed of cellulose. Although the fibril-like material visualized on the surface of protoplasts in many other systems is most likely cellulose, more careful characterization of this material is advisable before general 8 conclusions can be drawn about the occurrence and secondary structure of cellulose in the regenerating cell wall. 2. Kinetics of cellulose synthesis by culturedgprotoplasts. The onset of wall regeneration by protoplasts may vary according to the source of the material and cultural practices (Willison, 1976). Burgess gt al. (1978) reported that there was a lag of hours or days before fibrils could be detected on the surface of protoplasts isolated from tobacco meSOphyll cells and Antirrhinum leaves; they suggested that during this lag period the cellulose—synthesizing machinery was reassembled. Grout (1977) presented similar data for tobacco mesophyll protOplasts. In other systems, e.g. protoplasts isolated from cell cultures of Vicia hajastanna, fibril deposition was observed within minutes after the protoplasts were removed from wall-degrading medium (Williamson gt al., 1977). Asamizu gt al. (1977), reported that the cellulose content of carrot protoplast cultures gradually increased over eight days to a maximum level 50% lower than that of cultured cells. In a second paper, Asamizu and Nishi (1980) reported that when carrot protoplasts were supplied 14C-glucose, label accumulated in an alkali-insoluble a-cellulose fraction within five hours after the protoplasts were isolated. As noted previously alkali insolubility alone is insufficient proof of the presence of cellulose (Delmer, 1977). Thus while Burgess gt al. (1978) have proposed that freshly-isolated protoplasts are in general not competent to synthesize cellulose, there is evidence in some systems that there is no lag in microfibril biosynthesis during wall regeneration. 9 3. Other cell wall constituents. There have been a limited number of studies partially characterizing, on a biochemical level, the non-cellulosic components of the regenerating cell wall: soybean, Hanke and Norhtcote (1974); carrot, Asamizu and Nishi, (1980); and Vinca rosea: Takeuchi and Komamine, (1978); Tanaka and Uchida, (1979). The composition of the regenerating wall differed between and within the same experimental systems, but direct comparison between studies must be made with caution. Hanke and Northcote (1974), and Takeuchi and Komamine (1978), contrasted the composition and structure of the regenerating wall to that of the native cell wall. One dilenma is immediately apparent. What constitutes the regenerating cell wall? Conventional methods for the isolation of cell walls from intact cells depend on the huge size difference between the cytoplasmic organelles and pieces of the cell wall. Cell wall fragments may approach the dimensions of intact cells, so that they sediment rapidly at low centrifugal force, and are retained by coarse filters. During the early stages of wall regeneration, wall fragments are probably small and loosely organized. Growing cellulose chains are presumably anchored to the plasma membrane (Mueller gt al., 1980, Giddings, .££.21': 1980). However, during the early stages of wall regeneration in the absense of a preexisting cellulosic network, much of the newly-synthesized cell wall material may float away from the surface of the protoplasts (Hanke and Northcote, 1974; Prat and Roland, 1971). Furthermore, cultured plant cells are known to excrete polysaccharides and glyc0proteins into the culture medium, a characteristic likely to be shared by protoplasts. Thus during the early stages of wall regeneration it is difficult to separate cyt0plasm and cell wall, and 10 at the same time distinctions between the true cell wall constituents and extracellular materials are blurred. In addition, cell wall regeneration may not proceed synchronously; in any case the regenerating cell wall is likely to be a dynamic organelle. Thus, perhaps it is premature to build detailed models of the structure of the regenerating wall. Hanke and Northcote (1974) have studied cell wall formation by soybean protoplasts. Freshly-isolated protoplasts from suspension culture cells were supplied radioactive glucose in order to monitor newly-synthesized cell components after 20 or 40 hours in culture. After 20 hours in culture, protoplasts were not yet osmotically stable; the material retained on a sintered glass filter was designated the washed protoplast fraction, and the dialyzed filtrate was taken to represent both secreted material and cytoplasmic polymers. However, after 40 hours of culture the protoplasts did not lyse when collected on the glass filter. Therefore the filtrate consisted only of extracellular material. Comparisons were made between the two washed protoplast fractions obtained at the end of the culture periods, and also the corresponding dialyzed filtrates, although it is unlikely that these represented the same cytological cell fractions. Following 20 hours of culture, radioactivity from supplied glucose was incorporated into starch, protein, and 'cellulose' (determined by digestion with a crude cellulase preparation, obtained from the commercial product, Onozuka cellulase R 1500). One type of pectic polysaccharide was identified in the dialyzed filtrate; it was characterized on the basis of its electrophoretic mobility as a weakly acid pectinic acid, and contained some galacturonic acid. After 40 hours of culture, two 11 additional pectins were isolated from the dialyzed medium fraction. All three pectic polysaccharides had electrOphoretic characteristics similar to pectins isolated from the parent soybean callus, which also contains additional classes of pectins. The three pectins were not found associated with the washed protoplast fractions after either 20 or 40 hours of culture and the authors interpreted this to mean that wall regeneration by soybean protoplasts was aberrant because of the absence of synthesis of some pectins associated with the lack of assembly of other pectins into the developing wall. Cultural conditions required for the isolation of protoplasts may also affect the synthesis of cell wall polymers. Boffey and Northcote, (1975), studied pectin synthesis during the formation of a cell wall by plasmolyzed tobacco cells. Plasmolysis had altered the synthesis of some pectic polymers when compared to unplasmolyzed tissues. Incorporation of supplied 14C-glucose into galacturonosyl and rhamnosyl residues of the bulk pectin fraction decreased, while incorporation into arabinosyl residues was not affected or was only slightly enhanced. This was interpreted to indicate that the synthesis of individual pectic polymers is separately regulated. Asamizu and Nishi, (1980) examined the pattern of incorporation of 14C-glucose and 3H-myoinositol by carrot protoplasts into polysaccharide-containing polymers. In one set of experiments protoplasts were labeled for five hours after varying periods of culture (during which the wall was forming). The labeled protoplast samples were mixed with cultured carrot cells and then homogenized. Cell walls were isolated by conventional procedures using centrifugation and multiple washing steps. The walls were fractionated 12 by extraction into pectin, hemicellulose and an alkali-insoluble 'a-cellulose' fraction. As noted previously, alkali insolubility is not a unique property of cellulose. In samples from protoplasts cultured for a total of 5, 22, 46, 70 or 100 hours, radioactivity was found in all three classical cell wall fractions; however the proportion of radioactivity in pectin, hemicellulose or 'a-cellulose' fractions continuously changed. It is not clear whether the nascent cell wall from protoplasts cultured for only short periods, was isolated by these procedures. The overall neutral sugar composition of the regenerating walls from protoplasts cultured for 24 hours or seven days differed from that of the cell walls from seven day old cultured cells. Here a similar comparison of the extracellular, 80% ethanol-precipitable polymers from protoplasts also differed from that of material isolated from the medium of cultured cells. After 24 hours in culture, with continuous labeling from 14C-glucose, the distribution of radioactivity in neutral sugar residues was determined from the three cell wall fractions. Arabinosyl and glucosyl residues were the predominant radioactive sugars in the pectic fraction, (although glucose is not normally found in pectic polysaccharides from native cell walls). Radioactive glucosyl, arabinosyl and galactosyl residues were found in the hemicellulose fraction; both galactosyl and glucosyl residues contained appreciable amounts of label in the 'a-cellulose' fraction. This labeling pattern is generally similar to that obtained with carrot cells in suspension culture (Asamizu and Nishi, 1979). Thus these reports indicate that all of the classical components of the cell wall are synthesized during wall regeneration by carrot protOplasts. However, the data presented do not discriminate 13 between the possibilities that specific pectic or hemicellulosic polysaccharides are absent during some stages of wall regeneration and/or that the synthesis of Specific pectic or hemicellulosic polymers is not coordinated during wall regeneration as compared to during growth of the native cell wall. Takeuchi and Komamine (1978) examined the regenerating wall and extracellular polysaccharides from Vinca rosea protoplasts, isolated after three days of culture. Protoplasts were collected from the culture medium, washed, broken by homogenization, and the wall was isolated by conventional techniques. The regenerated wall of VIBES protoplasts was fully soluble in 24% KOH; thus it did not contain any crystalline cellulose. The native cell wall of Vinca does contain cellulose, pectins and hemicelluloses (Takeuchi and Komamine, 1978). Instead the regenerated wall was composed of 3- and 4-linked glucans which are also present in the native cell wall of [1393. They also examined extracellular polysaccharides from protoplasts and these were found to be similar to those in the medium of liquid suspension cultured cells; these polysaccharides included polyuronides and 3,6-linked arabinogalactan. However, these studies were not detailed enough to ascertain if specific cell wall pectins or hemicelluloses were not synthesized by protoplasts or were not assembled into the developing cell wall. C. Cell Wall Biosynthesis While cell wall regeneration provides one framework in which to examine wall biogenesis, a complementary approach involves elucidation of the enzymology of polysaccharide biosynthesis. Thus far only 14 limited information is available on this aspect of cell wall biology, (Delmer, 1977; Darvill gt al., 1980). Sugar nucleotides act as high energy intermediates, either directly, or possibly via lipid/and or protein-linked intermediates, in the synthesis of cell wall polysaccharides (Delmer, 1977; Karr, 1976). It is believed that pectic polysaccharides and hemicelluloses (with the exception of 3-8-glucan) are synthesized in the Golgi system (Ray ‘gt‘al., 1976; Robinson, 1977). In cell fractionation studies, an enzyme which utilizes UDP-glucose, to produce a 4-8-glucan, is considered a tentative marker enzyme for the Golgi system (Ray gt al., 1969). The enzyme, glucan synthetase 11, is believed to synthesize the backbone of the xyloglucan polysaccharide and a second enzyme (a UDP-xylose: polysaccharide transferase) assembles the side chains (Quail, 1979; Ray, 1980). Cytological observations suggest that the individual glucan chains of cellulose are synthesized at the plasma membrane and concomitantly assembled into microfibrils (Brown and Montezinos, 1976; Mueller ‘gt al., 1976). Glucan synthetase II, the 3-8-glucan synthetase (E.C.2.4.1.34) is also believed to be localized at the plasma membrane and is considered to be a provisional marker enzyme for the plasma membrane (Quail, 1979). Crude membrane preparations derived from a variety of species, have been used to study the synthesis of polysaccharides in vitro. One difficulty in studying glucan synthesis in vitro is that the only method of detection is the incorporation of radioactively-labeled glucose, from the nucleotide sugar, into an insoluble product 15 (Villemez, 1973). Delmer (1977) noted that far too often these insoluble products have not been adequately characterized, thus resulting in erroneous interpretations. Earlier studies claimed in vitro cellulose biosynthesis with membrane preparations from Phaseolus, or other species; these utilized either GDP-glucose or UDP-glucose to synthesize alkali-insoluble products (Barber 33 al., 1964; Brummond and Gibbons, 1965; Elbein 35 al., 1963; Franz and Meir, 1969). In most instances these results have been reinterpreted: GDP-glucose incorporation is stimulated by GDP-mannose (Villemez, 1973), suggesting that this probably represents the synthesis of glucomannan. UDP-glucose is utilized as a substrate for the synthesis of xyloglucan, 3-e-glucan, and/or mixed linkage glucans (in monocots). (Delmer, 1977; Ray, 1980; Smith and Stone, 1973). Although indirect evidence favors UDP-glucose as the most probable substrate for cellulose biosynthesis-13.1119, there is no convincing evidence of in vitro synthesis of crystalline cellulose in systems derived from higher plants (Darvill 33 al., 1980; Delmer, 1977). Carpita and Delmer (in press) have speculated that there is some crucial role of plasma membrane integrity required for cellulose biosynthesis which would be largely destroyed during membrane isolation. Another consideration in the study of polysaccharide biosynthesis is, the extent to which compartmentation controls the types and complexity of polysaccharide synthesized in vivo (Villemez, 1973). Most of the work on polysaccharide synthesizing systems from higher plants have utilized crude particulate systems. The same nucleotide sugar, uridine diphosphate glucose serves as substrate, for more then 16 one polysaccharide-synthesizing enzyme. Furthermore it is difficult to identify the products of such in vitro systems and distinguish which polysaccharide-synthesizing system they arise from in vivo. ? xyloglucan :‘fii:? glucomannan UDP-glucose —-o —-—, _.? 3- & 4- B -glucan \? cellulose ? 3-B-glucan None of the cell wall polysaccharide-synthesizing enzymes from a higher plant have been isolated and extensively characterized. 0. Regulation of Biosynthesis The rates of synthesis of cell wall polysaccharides change in response to various factors, including: 1) plant growth factors (e.g. indole acetic acid, Evans, in press; Spencer gt al., 1971); 2) plasmolysis (Boffey and Northcote, 1975); 3) stage of development (Meinert and Delmer, 1975); 4) wounding (e.g. instaneous induction of 3-e-glucan synthesis: Brett, 1978); 5) temperature stress (Smith and McCully, 1977). Some information is available about the underlying mechanisms which may regulate the synthesis of cell wall polysaccharide: For example, during development of the cotton fiber, the cellular concentration of UDP-glucose increases, concomitant with the maximum rate of cellulose biosynthesis. (Carpita and Delmer, in press). Indirect evidence suggests a role for IAA-induced 17 transcriptional/translational control of polysaccharide-synthesizing enzymes during indole acetic acid-induced growth (Evans, in press). As indicated previously, the 3-3-glucan synthetase and cellulose synthetase are influenced by membrane integrity, disrupting the plasma membrane activates the 3-a-glucan synthetase while eliminating cellulose synthesis (Carpita and Delmer, in press). How is the activity of the 3-8-glucan synthetase rapidly modulated? The orientation of the 3-3-glucan synthetase within the plasma membrane, periplasmic versus cytoplasmic, could control the utilization of endogenous substrate (HOP-glucose) and thereby regulated the synthesis of glucan. Three models illustrated below suggest how the orientation of the enzyme within the plasma membrane, might regulate enzyme activity. A. l'p glucan lb uoe'c B. awe C. U090 18 The first model proposed by Hughes and Gunning (1979) shows the active site of the enzyme oriented toward the periplasmic face of the plasma membrane. Supplied USP-[14CJ-glucose it utilized to synthesize 3-e-glucan. Cytoplasmic pools of UDP-glucose do not serve as substrate, except when the cell is wounded or stressed, allowing the UDP-glucose to leak thru the plasma membrane to the enzyme. In the second model, favored by Mueller and MacLachlan (1980) the plasma membrane is slightly permeable to the supplied UDP-[14CJ-glucose; as the nucleotide sugar crosses the plasma membrane of the protoplast or intact cell, it is immediately utilized by the 3-e-glucan synthetase which has a high affinity for the nucleotide sugar. However, this model does not explain how the activity of the enzyme is regulated such that the cytoplasmic pools of UDP-glucose are not utilized for the synthesis of 3-8-glucan. The third model, suggested from a report by Christopher Brett (1978), implies that the 3-a-glucan synthetase is normally in some latant form, perhaps buried within the plasma membrane. At certain stages of development or particularly in response to stress or wounding, the enzyme is activated. This thesis addresses questions pertaining to cell wall biogenesis. The first chapter investigates cell wall regeneration by soybean protoplasts as a model for cell wall synthesis, secretion and assembly. The second chapter deals with preliminary studies to isolate polysaccharide-synthesizing enzymes, in order that the biochemistry of these enzymes may be better characterized. Finally chapter three examines how one of these enzymes, the 3-s-glucan synthetase may be regulated in situ, both as a function of its orientation within the 19 plasma membrane, and in response to perturbations in its physical environment in the membrane. MATERIALS AND METHODS Chemicals and reagents D-[(U)-14C]-glucose and UDP-[(U)-14C]-glucose were obtained from New England Nuclear (Boston, MA, USA) or from ICN Pharmaceuticals (Irvine, CA, USA). A Streptomyces cellulase preparation and a 3-a-endoglucanase preparation from Rhizpous were the generous gifts of Dr. E.T. Reese, (U.S. Army Laboratories, Natick, MA, USA). Porcine pancreatic a-amylase (Type I-A), Commassie Brilliant Blue G, concanavalin A (IV), cyanogen bromide activated Sepharose 6B, Sepharose 28, Reactive Blue Agarose, bovine serum albumin, valinomycin, cholic acid and Ficoll 400 were purchased from the Sigma Chemical Corp. (St Louis, MO, USA). Ficoll was purified before use as described in Boller and Kende (1979). Cholic acid was recrystallized twice from 70% ethanol. Nigericin was obtained from Eli Lilly and Co. (Greenfield, IN, USA). The herbicide DCB, (casoron), was a gift from Thompson-Hayward Chemical Co., (Kansas City, KA, USA). Ultragel ACA 34 was purchased from LKB Instruments (Rockville, MD, USA). Sephadex G-25 was obtained from Pharmacia Fine Chemicals (Piscataway, NJ, USA). Commassie Blue protein reagent and Bio-gel P-6 were purchased from BioRad Laboratories, (Richmond, CA, USA). Polyethylene glycol 4000 (PEG 4000, "Baker" grade) was obtained from T.T. Baker Chemical Co., (Phillipsburg, NJ, USA). Pentaerythritol is produced by Pfaltz-Bauer (Stamford, CN, USA). All other reagents were of analytical grade. 20 21 Double distilled, deionized water was used for all solutions. However, on at least four occasions over the last three years, the liquid-suspension cultures gradually deteriorated: two and three day old cell cultures were noticeably brown, indicating that the cells were producing phenolic compounds. The accumulation of phenolic compounds is usually associated with the stationary growth phase or stress (Hahlbrock and Griesebach, 1979). Simultaneously protoplast yields would decline. This was usually correlated with an accumulation of volatile amines in the double distilled H20 supply used to prepare culture medium. Cotton ovule, Dictyostellium and mammalian cell cultures are adversely affected as the volatile amines accumulate (Ken Poff, Justin McCormick, personal communications). The amine problem was circumvented by changing the ion exchange system of the primary distilled H20 supply at frequent intervals: These ion exchange cartridges gradually deteriorate then leach quaternary amines in the distilled H20; furthermore, when the cartridges detoriate to this stage the system no longer removes morpholine, a volatile amine and an antiscale agent in the steam supply, from the distilled water. Cell wall degrading enzymes were partially purified, as described by Gamborg (1975), with minor modifications. Cellulase R-IO (Onozuka; Kinki Yakult, Mfg Co., Ltd. Nishinomiya, Japan), Macerase (Calbiochem, Irvine. CA, USA) or Cellulysin (Calbiochem, were each dissolved in cold water, (10 to 15 g/100 ml) and clarified by centrifugation. The supernatant was applied to a Sephadex G-25 column (90 x 5.5 cm) and eluted with water. Fractions were monitored for protein with Bradford's reagent (Bradford, 1976); those which contained protein were 22 retained and lyophilized. The resulting material (ca. 10 to 40% by weight of the commercial preparation) was used in protoplast isolations. Cell Cultures A liquid suspension of cells of Glycine max L. Merr cv. Mandarin, designated SB-I (derived from root tissues), was obtained from Dr. Gerhard Weber, (University of Utah, Salt Lake City, UT, USA). Cultures were maintained in 53 ml of 185 medium, (Gamborg, 1975) in 125 ml Erlenmeyer flasks, at 27°C in the dark on a gyratory shaker at 125 rpm. The cells were subcultured during log phase growth by a 1 to 3 dilution at 3-4 day intervals. The cell line was also maintained as a callus culture on medium solidified with agar; calli were subcultured monthly. On at least three separate occasions calli were used to start fresh suspension cultures. Protoplast Isolation All manipulations were performed under sterile conditions in a laminar flow hood. Two day old suspension cultures were transferred to conical tubes and allowed to settle under gravity. The cells were washed twice with a solution containing 85 salts and vitamins (Constabel, 1975). One volume of 3% (w/v) Cellulysin (or Cellulase) and 1% (w/v) Macerase in 10% (w/v) sorbitol, pH 5.5 in water, was added to the cells. The suspension of cells was transferred to a Petri dish (100 x 20 mm), and was incubated for 2.5 h on a reciprocal shaker (ca. 30 cycles min-1), at 27°C, in the dark. 23 The suspension was filtered through Nitex Cloth (linear pore size, 48 um; Tabler, Ernst and Traber, Inc. Des Plaines, IL, USA), then protoplasts and cell debris were collected by centrifugation (8 min, 120 x g in a clinical centrifuge). The protoplasts were purified by flotation upward through a ficoll gradient in a manner similar to that described by Boller and Kende (1979): First the pellet containing protoplasts and debris was resuspended in 6 to 7 ml of 19% Ficoll (w/w in wall regeneration medium, see below), and placed in the bottom of a 12 ml centrifuge tube. This suspension was overlain with 2 ml each of 16% and 13% ficoll and finally with wall regeneration medium. The gradient was centrifuged for 20 min at 170 x g, at room temperature in a clinical centrifuge. Protoplasts were collected from the interface between 13% ficoll and wall regeneration medium. At this stage, the preparation appeared to contain only protoplasts; no intact cells or wall debris could be detected by routine light microscopy. Damaged and lysed protoplasts were trapped in the denser Ficoll layers. Typically 20 ml packed cell volume yielded betwen 1 x 107 and 4 x 107 protoplasts. Protoplasts were washed three times with wall regeneration medium and collected each time by centrifugation (3 min, 120 x g). Protoplasts were resu5pended at final concentrations between 3 x 105 and 2 x 106 ml"1 in wall regeneration medium supplemented with the appropriate labeled sugar; 3 to 5 ml samples of protoplast suspension were transferred to individual plastic Petri dishes (60 x 15 mm) for incubation. Protoplast wall regeneration medium is that described by Constabel (1975) for soybeans, except glucose was omitted and replaced with an equimolar amount of sorbitol. 24 To increase the reproducibility of protoplast yields to a level adequate for routine experiments, the procedure for protoplast isolation was eventually modified as follows: i) Cell suspensions were subcultured 18 to 24 h prior to use. ii) The wall degrading medium was altered to include 30mM Na-MES, pH 5.5, and SOmM mercaptoethanolamine (Tom Boller, personal communication). This modified protocol was used to prepare protoplasts for the experiments described in the Section A and B3 of Chapter One and for all of the experiments in Chapter Three. Measurement of Water Potential The water potential of protoplast culture medium was measured on a Wescor Dewpoint Hygrometer (Wescor, Inc., Logan, UT, USA) which had been calibrated with standard solutions of sucrose (Nelsen gt al., 1978). The water potentials of various dilutions of a concentrated PEG 4000 solution were determined; these data was used to calculate the appropriate dilutions of the PEG 4000 stock solution to prepare protoplast media at -11, -13.5, -16 bars (see Table 1-1). Similar measurements and calculations were performed to prepare the sorbitol- based medium. Isolation of Polysaccharides and Glycoproteins from Cell Walls and Spent Medium of Liquid Suspension Cultures Two day old suspension-cultured cells were collected on a sintered glass filter. Three and one half volumes of ethanol were added to the spent medium (filtrate) and the mixture was chilled at 4°C overnight. The precipitate, containing polysaccharide and protein, was washed four , times with 70% (v/v) ethanol. This material resembles the secreted 25 extracellular polysaccharide (SEPS) of cultured sycamore maple and secreted arabino-galactan protein described by Lamport (1977, 1978). Cells were washed extensively with a solution of BS salts (at equivalent concentration to that in the culture medium; ca 500 ml per 20 9. cells). The cells were resuspended in cold 100 mM potassium phosphate buffer, pH 7, with 4 mM sodium metabisulfite and disrupted at 4°C by three, 2 min cycles of sonication with a Biosonic VI sonifer (Bronwill Scientific, Rochester, NY, USA). Cell walls were prepared as described by Talmadge gt al. (1973). Following the final acetone wash, the pellets were washed in 50% methanol three times and once in water. Walls were resuspended in 5 ml of 50 mM potassium phosphate buffer, pH 6.8, (ca. 10 mg ml'l) with 100 U a-amylase per ml of suspension and incubated with 50 ul of toluene for 25 h at 30°C in a gyratory shaker (ca. 125 rpm). The walls were washed with water three times and once with acetone, lyophilized, and stored in a vacuum desicator. Isolation of Newly-Synthesized Polymers For the time course experiments (Chapter one; Figs. 1 thru 4), incubations were terminated by the addition of 4 volumes of ethanol to give a final ethanol concentration of 80% (v/v). In other experiments protoplasts were collected by centrifugation (3 min, 120 x g); and were washed four times with cold wall regeneration medium then resuspended in 80% ethanol. This was designated the protoplast fraction. The first supernatant (spent wall regeneration medium), was brought to 80% ethanol and the precipitate, consisting primarily of polysaccharide and protein, was collected by centrifugation (10 min, 10,000 x g). This was designated the extracellular fraction (ECM). The various 26 precipitates were washed four times with 80% ethanol and with chloroform methanol (1:1, v/v). To measure the radioactivity in soluble cytoplasmic pools, washed protoplasts were disrupted in 80% ethanol and chilled overnight. The precipitate was collected by centrifugation and the first supernatant was retained: this constituted the 80% ethanol-soluble cytoplasmic contents. Cellulose Content The cellulose content of cell walls was determined by the method described by Updegraff (1969). Determination of the Radioactivity in Crystalline Cellulose Carrier cellulose (ca. 0.5 mg) was added to the ethanol precipitate and the sample was boiled in 2 ml of acetic-nitric acid reagent (AN reagent, Updegraff, 1969) for 60 to 90 min. Water (3 ml) was added to reduce the viscosity and the insoluble material was collected by centrifugation (10 min, 10,000 x g). The supernatant was carefully removed and the pellet was washed four times with 5 to 10 ml of water. For direct determination of radioactivity the pellet was dissolved in 10 ml Biosolve (Beckman Scientific, Irvine, CA, USA) or ACS (Amersham, Arlington Heights, IL, USA) scintillation cocktail. Neutral Sugar Analyses The neutral sugar content of cell walls and the distribution of radioactivity in neutral sugar residues of the ethanol-precipitable material was determined after hydrolysis in 2N Trifluoroacetic acid at 120°C for 90 min. Crystalline cellulose is not readily hydrolyzed by 27 this procedure. Neutral sugars were reduced and acetylated by the procedure of Albersheim gt al. (1967). Following acetylation, sugar derivatives were redissolved in dichloromethane and analyzed by GLC. Separations were performed in a Hewlett Packard model 8350 gas chromatograph (Avondale, PA, USA) equipped with a variable effluent stream splitter and integrator. Nitrogen, was used as a carrier gas (22 ml min‘l). The glass columns (180 x 0.2 cm i.d. configuration 8), were packed with Supelco Support SP 2340 (Bellefonte, PA, USA). The alditol acetates were separated with temperature programming at 6° min‘l, from 140°C to 225°C. Acetylated sugar derivatives of the cell wall were identified by relative retention time. Radioactivity in samples derived from protoplasts was identified by coelution with alditol acetates of a standard sugar mixture: 50% of the effluent stream went to the flame ionization detector, and the remainder was diverted to a heated collection port. The alditol acetates condensed in Uniform Drop Size Pipets (Scientific Products, Romulus, MI, USA) fitted to the collection port; the condensed material was rinsed into vials with toluene-based scintillation cocktail (4.2 g PPO, 52.5 mg POPOP l‘l) and radioactivity was determined by scintillation counting. Methylation Analyses The linkages of (radioactive) neutral sugar residues were determined by methylation analysis according to the method of Hakamori (1964), as described by Maltby gt al., (1979) with the following modifications: After methylation samples were transferred to screw cap culture tubes (15 x 150 mm, with Teflon lined caps). Chloroform: 28 methanol, (2:1, v/v, 7 ml) was added to each, then 10 ml of water were added to extract unreacted methyliodide. The extraction was repeated seven times; each time the upper phase of methanol and water was removed, but any insoluble material at the interface and lower layer were retained. After the extractions, the lower layer and interface were transferred to a conical microflex tube, (Kontes, Vineland, NJ, USA), and dried under nitrogen. After acetylation, the remaining acetic anhydride was evaporated under nitrogen as an azeotrope with toluene, and the gelatinous residue was washed once with toluene. Dichloromethane, 0.5 ml, was added to each sample; the suspension was resuspended and centrifuged, in a bench top centrifuge (1 min, 625 x g) and the supernatant transferred to a clean vial. Chromatography was done on a Varian Aerograph GLC, (model 2100, Louisville, KY, USA) equipped with an effluent stream splitter (fixed ratio 10:1, collector to flame ionization detector). Helium was used as the carrier gas (30 ml min‘l). Separations were performed on glass columns (180 cm x 0.2 cm i.d.), packed with Gas-Chrom 0, (100-120 mesh, Applied Science Laboratory Inc., State College, PA, USA) coated with a mixture of 0.2% poly(ethylene glycoladipate), 0.2% poly(ethylene glycol succinate) and 0.4% silicone XE-lISO. The alditol acetates of permethylated sugars were separated with temperature programming (115°C, with a 5 min hold, then 4° min"1 to 190°C). Fractions were identified by coelution with known sugar derivatives and the effluent was collected in tips of Pasteur pipets (9 inch, Scientific Products, Romulus, MI, USA). The identify of permethylated sugar derivatives of the cell wall and extracellular polysacharrides was verified by combined gas chromatography-mass spectrometry of samples which were reduced with 29 sodium borodeuteride; analyses were performed on a Hewlett Packard gas chromatograph-mass spectrometer model 5985A at the MSU mass spectrometry facility. Radioactivity was determined as described above. In some experiments separations were carried out on a Hewlett Packard model 8350 gas chromatograph, with nitrogen as a carrier gas (22 ml min’l); the elution profile of standards was similar on both chromatographs. Cell Counts The average number of cells per 0.5 ml packed cell volume was determined as follows. Duplicate 10 ml aliquots of cell suspensions were allowed to settle under gravity in 15 ml graduated conical tubes. The supernatant was removed and the cells were resuspended in approximately 0.25 ml concentrated sulfuric acid (as suggested by Dr. R. Greisbach). The suspension was diluted to 10 ml with H20 and cell clumps were disrupted by passage thru a syringe equipped with a 22 1/2 gauge needle. The acid treatment was repeated if necessary. The average cell number per ml of suspension was based on the average of four individual determinations using a hemacytometer. 0.5 ml packed cell volume contained approximately 1.1 X 107 cells. Protein Determinations The protein content of samples was determined using the method of Bradford (1976). Samples (0.1 ml), containing 10 to 100 ug of protein were mixed with 5 ml of Commassie Brilliant Blue G reagent and the 3O absorbance was monitored at a wavelength of 595 nm. Bovine serum albumin was used to prepare a standard curve. 1. Protein content of soybeangprotoplasts. The protein content of protoplasts was measured based on a unit of 1 X 106 protoplasts (as determined by counting protoplasts on a hemocytometer), and was 0.35 mg per 106 protoplasts. 2. Samples in cholate buffers. Prior to assaying protein content, samples which contained cholate, were extracted with four to nine volumes of absolute ethanol at 70°C for 10 min. Cholate, which interferes with the protein assay, is soluble in hot ethanol. The precipitated protein was collected by centrifugation and washed with ethanol (1 X 1 ml). The precipitate was redissolved in 16.5% glycerol, 50 mM K°TES, pH 7.8, 400 mM sorbitol, 5 mM M9804 and protein content was determined using the micro method described by Bradford, (1976). Bovine serum albumin, treated in a similar manner was used as a standard. Preparation of Samples for Electron Microscopy Prot0plasts were cultured for the times indicated in the figure legends, then resuspended in 1% (w/v) glutaraldehyde in wall regeneration medium at room temperature as described in Fowke gt al., (1975); Williamson gt al., (1976). After 1-2 h the protoplasts were resuspended in 3% glutaraldehyde in the same medium and held overnight at 4°C. The samples were then washed in 0.05 M sodium phosphate buffer and postfixed 2 h in 1% osmium tetroxide in 0.05 M sodium phOSphate, pH 6.6 at 4°C. After 2 washes in cold distilled water, samples were dehydrated by passage through a graded ethanol series followed by amyl 31 acetate. Samples were transferred to poly-L-lysine coated cover slips and allowed to dry as described by Mazia gt al., (1975). i The samples were shadowed with platinum-carbon at a 45° angle and then carbon coated at a 90° angle in a Balzers BA 360 vacuum evaporator. The replicas thus formed were removed from the coverslips by slowly dipping them into 50% hydrofluoric acid, rinsed by transferring them through two changes of distilled water, and cleaned by floating them on sodium hypochlorite (bleach, available commercially) for 1-3 days. The cleaned replicas were mounted on uncoated 300 mesh copper grids and examined in a Siemens 1A electron microscope. Preparation of Membrane Bound Glucan Synthetases from Soybean Cells Unless otherwise stated, the protocol used for preparing soybean cell membranes was as summarized in Fig 2-4 and described below. Suspension grown cells (2d) were collected by filtration, in a Buchner funnel lined with Miracloth (Chicopee Mills, Inc. Milltown, NJ, USA). The cells were washed extensively with ice cold H20, then weighed. The cells were then plasmolysed in ice cold buffer (1.5 vol. per g fresh weight cells): 50 mM K-TES, pH 7.2, 400 mM sorbitol, 5 mM MgCl2, 5 mM DTT, 1 mM PMSF. The suspension (20 to 60 ml) was injected into the sample chamber of a Ribi Press (Sorvall Inc., Norwalk, CT, USA) and disrupted under pressure (Ca. 8000 PSI) while maintaining the temperature of the Ribi valve at 5-12°C. The brei was collected in a beaker on ice. The sample chamber and Ribi valve were flushed with buffer (10 ml) and this was pooled with the broken cells. 32 This treatment resulted in uniform cell breakage, but cell walls and other organelles remained recognizable under the light microsc0pe. The brei was filtered through Nitex cloth (linear pore size, 40 um) to remove large wall fragments. The filtrate was centrifuged at 500 x g at 5°C for 5 min. The pellet consisting primarily of small wall fragments was discarded. The supernatant was centrifuged at 30,000 x g, at 5°C, for 45 min. The resulting pellet constituted the membrane bound glucan synthetase preparation. Solubilization of the Glucan Synthetases The membrane pellet was weighed and resuspended in ice cold buffer (50 mM K-TES, pH 7.8, 1 mM M9504, 1 mM DTT, 1 mM PMSF) containing 30 mM BTP:cholate (at a ratio of 1 to 2 ml per 100 mg wet weight of pellet). The particulate suspension was disrupted in a Dounce homogenizer (10 strokes), followed by brief sonication (15 to 30 sec.) in a bath type sonifier. The suspension was stirred for 15 min. at 4°C to facilitate solubilization, then centrifuged at 30,000 x g, at 5°C, for 45 min. The resulting supernatant (second 30,000 x g supernatant) constitutes the solubilized glucan synthetase preparation. Glucan Synthetase Assay Reaction mixtures contained 150 to 190 pl of enzyme sample and 0.1 mM UDP-14C-glucose in a final volume of 0.2 ml in disposable glass test tubes (13 x 100 mm). The specific activity of UDP-glucose was either SuCi/umole or 10 uCi/umole. Unless otherwise stated incubations were for 20 min. at 25°C. Reactions were terminated by the addition of 0.6 ml of unlabeled UDP-glucose (1.2 mM) followed immediately by the 33 addition of 1.4 ml of absolute ethanol. A small amount of carrier cellulose was added to each tube (ca. 0.5 mg) and the samples were heated at 70°C for 15 min. to facilitate solubilization of lipids. Subsequently the samples were stored at -20°C for Z 3 h to aid precipitation of glucan products. Insoluble products were collected on glass fiber filters (2 cm. Whatman GFA or 934 AH, Whatman, Inc., Clifton, NJ, USA) on a 12 port Millipore vacuum filtration unit (Millipore Corporation, Bedford, MA, USA). The samples were washed with ice cold 70% ethanol (10 ml x 4) and chloroform methanol, (1:1, v./v.; 10 ml x 1). The filters were transferred to glass scintillation vials, dried, and radioactivity determined by scintillation counting in toluene-based cocktail (42 g ppo, 52.5 mg POPOP 1-1). When the composition of glucan products was to be further analyzed, the reactions were carried out in 15 ml Corex tubes (Corning Glass, Corning, NJ, USA). The ethanol-precipitated products were collected by centrifugation (10 min, at 10,000 x g) and washed sequentially with 70% ethanol (5 ml x 4) and chloroformzmethanol (1:1, 5 ml x 1). Separation of Membrane Vesicles on ConA Sepharose 1. .Preparation of concanavalin A Sepharose 68. The affinity gel was prepared by the method of Gurd and Mahler (1974) with some modifications as follows (C. Caldwell, personal conmunication): ConA was dialysed overnight, at 4°C, against 100 mM sodium carbonate buffer, pH 8.4, with 200 mM awnethylmannoside. This buffer and all others used in subsequent steps contained: 1 mM MgCl2, 1 mM MnClZ and 1 mM 34 CaClz. Cyanogen bromide-activated Sepharose 6B was hydrated for 3 h in 1 mM HCl. The activated Sepharose was mixed with conA (ca. 25 mg lectin to 1 gram of gel) in 100 mM sodium carbonate buffer, pH 8.4, (10 ml buffer per gram gel), and incubated overnight at 4°C with gentle agitation. The coupling reaction was st0pped by the addition of ethanolamine (final concentration 1 mM) and the mixture was incubated for one additional hour. The conA-Sepharose beads were washed exhaustively with: 1) 100 mM Na-carbonate buffer, pH 8.4, 100 mM NaCl, 750 ml 2) 100 mM Na-Acetate buffer, pH 4.0, 100 mM NaCl, 750 ml 3) water 750 ml and 4) 10 mM K-HEPES, pH 7.2, 5 mM MgCl2, 1 mM Mn Clz, 10 mM CaClz, 400 mM sorbitol, 750 ml. The affinity gel was stored in this buffer (4) with 0.02% sodium azide at 4°C. 2. Preparation of membrane vesicles and affinity chromatography. Protoplasts (2-4 x 107) were disrupted by osmotic shock in 2 ml of ice cold buffer: 20 mM K-HEPES, pH 7.2, 10 mM MgCl2, 20 mM CaClz, 2 mM MnClz. After 2 min., the water potential of the solution was lowered by the dropwise addition of 2 ml of 800 mM sorbitol containing 2 mM DTT and 2 mM PMSF, with continuous mixing. An aliquot of the suspension was withdrawn to determine total glucan synthetase activity and the remainder was centrifuged at 10,000 x g for 15 min. The resulting membrane pellet was resuspended in a Dounce homogenizer (10 strokes), in 2 ml of column buffer containing: 10 mM KoHEPES, pH 7.2, 400 mM sorbitol, 5 mM MgCl2, 10 mM CaClz, 1 mM MnClz, 1 mM PMSF, 1 mM DTT. An aliquot was removed to determine glucan synthetase activity and the remainder (1.2 ml) was applied to a 2 ml conA Sepharose column. After 20 min, vesicles were eluted with 16 ml of column buffer, at a rate of 4 ml-hr'l. Subsequently, 1.2 ml of 35 buffer, supplemented with 200 mM a-methylmannoside, was applied to the column. After 10 min, the putative right-side-out plasma membrane vesicles were eluted with 10 ml of column buffer (with a-methylmannoside) at a rate of 6 ml-hr‘l. Finally the buffer was supplemented with 30 mM BTP-cholate, (final pH 7.8) and residual protein and lipid were eluted from the column (10 ml of cholate buffer). Fractions containing approximately 0.5 ml were collected and monitored for absorbance at 280 nm light. Fractions containing vesicles (A280>0.20) were pooled (Fig. 3-2) and assayed for glucan synthetase activity. Induction of Transmembrane Potentials Across Vesicles Derived From Soybean Protoplasts Freshly isolated soybean protoplasts were disrupted by osmotic shock by resuspension in ice cold 10 mM BTP-SO4, pH 7.4 (or BTP-MES, pH 7.2), 1 mM DTT, 3 mM M9304. Membrane vesicles were formed by the gradual addition of six volumes of 60 mmolal PEG 4000 (in the same buffer) (Carpita and Delmer, in press). The vesicles were divided into aliquots (200 - 1500 99 protein per sample). Concentrated salt solutions and ionophores were added to the samples (see Fig. 3-4); these combinations of cations and anions with different relative permeability coefficients, are believed to induce transmembrane potentials across the vesicles (Bacic and Delmer, in press; Pressman, 1976). 36 UDP-glucose was supplied at 0.1 mM final concentration; the specific activity of the label and reaction times are indicated for each experiment. Estimated Counting Efficiency of Radioactive Samples Standard liquid scintillation quench curves, utilizing an external standard or H value (Beckman instruments), were used to determine counting efficiency. The counting efficiency for samples of labeled polysaccharide on glass fiber filters cannot be determined by standard methods because the counting geometry for such samples is not known. For these samples, counting efficiency was estimated by spotting a standard amount of 14C-glucose or 14C-aspartate on the filters and then counting these samples in toluene-based scintillation cocktail. The estimated counting efficiency on Whatman GFA filters was 80%; that of Whatman 934AH filters was 63%. CHAPTER 1. EARLY EVENTS IN CELL WALL REGENERATION Results A. Incorporation of 14C-Glucose In order to monitor the synthesis of polysaccharides during wall regeneration, protoplasts were cultured in the presence of 14C-glucose at high specific activity and low concentration. Incorporation of label into total 80% ethanol-precipitable polymers is enhanced by decreasing the concentration of solute (sorbitol or PEG 4000), possibly because this increases the water potential of the culture medium (Table 1-1). However, at any given water potential the level of incorporation of [14CJ-glucose is influenced by the solute used (c.f. sorbitol with PEG-4000). The water potential and solute composition of the medium also affect the distribution of label incorporated into cellulose (see below) and other 80% ethanol-precipitable polymers. Other factors, including the general physiological state of the protoplasts and the density at which they are cultured may also influence the absolute magnitude of incorporation (c.f. the values for incorporation in PEG-based medium at -11 bars, Table 1-1, protoplast density 7 x 105 mi-l, with that of o uMDCB, in Table 1-2, protoplast density 12 x 105 ml'l; also see Rubin and Zallin, 1976). These factors varied between experiments; there- fore, the composition of the culture medium and the protoplast density are noted for each experiment described in the following sections. 37 38 In order to conveniently monitor the synthesis of polysaccharides, individual samples generally contained at least 2 x 106 protoplasts. Thus, roughly 1 x 107 protoplasts are required for a small experiment. Under optimal conditions, 20 ml packed volume of cells yield 1 to 4 x 107 protoolasts. However, these yields are drastically reduced by any condition which adversely affects the growth rate of soybean cells: power failure, change in culture room temperature, and particularly the accumulation of amines in the water _supply used to prepare culture medium. It is not feasible to scale up by increasing the amount of starting material; the cost of cell wall-degrading enzymes is prohibitive. Instead this problem was remedied by minor modifications in cultural procedures and rigorous precautions to maintain rapidly growing liquid suspension cultures. These procedures are described in more detail the Materials and Methods. B. Cellulose Synthesis 1. Cellulose content of the cell wall of cultured soybean cells. Cell walls were prepared from rapidly growing liquid suspension cells harvested one or two days after subculture. Ona dry weight basis, the average cellulose content of one day old walls is 18% and that of two day old walls is 22%. These values are similar to the amount of cellulose found in the cell walls of cultured dicots (Darvill gt al., 1980; Albersheim, 1976). Cellulose is a significant fraction of the cell wall; experiments described below examine cellulose synthesis as a component of cell wall regeneration. 39 .—o:e;um we mmE:_o> csod do :owu_vve on» an umue=_5cmu mew: wee ; H to; mczuecmaEmu soot we use newccmu mew: m:o_uen=o:H .m_oE=\_u: oH do xup>wuoe owu_umam m :u_3 .ZEH do :o_uecucwucou .ecpm a m>wm op cm_paa:m we: mmoozpmimuefiu .medza cw mape_ose ooHv ooe owe new A_e_oae oowv —ou_ncom umuecacmocoo do mco_uzpwc muewcaocaaw to; mm>c=u uceuceum soc cm:P5cmumu mow cmueo_ucp we _e_u=muoa swam: m ;u_3 .ooce owe co pou_acom sup: cmucmempaaam . _omu :5 m.o . ommz 25m .m.m In .muz.m_c» zeofiucwwmzn :P umucmamsmmc mum: A_E m :P moH x ~.~v mummpaouocm HiH open» 40 .z can mama—aoaoca moH can emuecoqcoucv mmouzpmimo .a_cms menu—=9 ecu do _e_u:maoa Lowe: peep; one we ummmmcaxm we wH mmueo_—a=u you H mmpoea we nmmmmcho mm:_e>n mus—om do mcopueeucmucoo ache so“ oe¢.~ mac. mNH mmH oqo.~ oew.~ Ha- ~.om omH.N eeo. mod o.mHH omm.~ oom.~ m.m~- oooe mum o.mm omo.~ emo. me «.mm cam.“ can.“ 0“. mom oesdma owe. 5mm mum oom.mfl ooo.e~ HH- mom om~.m mmo. mmm umw o~m.m om~.m m.m~- fled cmm.e _ee_eeem «no. wad Now om~.m om~.m oHi ¢.me o~¢.m ado. e.ee c.5e owe.m cme.m e3- mcmExAOQ «wueam Amcmsx_oa peuou\mmo—=__mov o_mo—:—qu-co: —m_ucmuoa cm~Pppooum 952 o omega: 33:28 0 omega £33 a Sun: ofiosmo .mumm—aouoca somehow x5 mcmexpoa mpamu_a_omcai—ocmgum cacao new mmo_=—_mo ou:_ mmouz—m do :c_uecoacoocw mg» co :owuocucmocou owe ecu —ouwncom do wooded use HIH mpnmh 41 2. Chemical evidence that cellulose is synthesized during the first hours of wall regeneration. Polymeric constituents of plant cells with the exception of cellulose, are degraded when treated at 100° for 30 to 60 min. with acetic acid, nitric acid and H20 (8:2:1, v/v, AN reagent; Updegraff, 1969). Crystalline cellulose is believed to be resistant to AN reagent, although low molecular weight 4-3-9lucan chains may be hydrolyzed under these conditions. The AN treatment was utilized in labeling studies with protoplasts to measure cellulose synthesis during regeneration. AN resistant material represents a very small fraction of the labeled material (Table 1-1). However, it was first necessary to confirm the validity of this procedure. The following analyses were performed on samples of labeled AN-resistant material derived from total 80% ethanol-precipitable polymers isolated from protoplasts which had been supplied [14CJ-glucose: a) AN-resistant material was hydrolyzed and the resulting mono- saccharides separated by descending paper chromatography (Saeman ._£.El-, 1963). More than 80% of the radioactivity in the hydrolysate in monosaccharides co-chromatographed with the glucose standard (Fig. 1-1). b) Another sample was degraded overnight with a partially purified cellulose preparation from Streptomyces (as described in Heiniger and Delmer, 1977). Cellobiose and glucose in an approximate ratio of 3:1 were the predominant small radioactive saccharides released (Fig. 2). Cellobiose, a disaccharide, is the repeating unit of cellulose, thus confirming that the AN-resistant material contains cellulose. Free glucose is the product of an enzyme contaminant(s) in 42 .mmo_xx "—zx .mmo:_aecm ”use .mmooz—m "zpm .omouom—em "—mm .mmownoF—mu "mu .m:_u:=oo covueppwu:_um as» An umcwEmemu »u_>_uueo_uec do co_u=a_cum_v any new ma_cum 50 H ou:_ use we: Emcmoueeocgu on» .A>\> .Humuwv om: "m:_uwcxa ”mumumom .zsum cw umao_w>ou .xgnecmoumeoczo cmaea mc_c=mummu x9 cmumccgmm mew: mucmuceum mamwcgocqae can mmupcmzooemocoe mcpupammc ms» .Amooav .Hm.mw.=mamem :_ umn_commu mm cwue o_c=$_:m ;u_3 vmmapocc»: we: .awcmums accumwmmc iz< esp .z mg cow mmou:_mumueflu gaw: umumnau:_ zoom on: sows: mumepaouoca Eocw cm>_cmv .mcmsapoa m—neu_a_omcau_ocm;um pcuop soc» emceawcq we: pe_cmuea ucaumwmmcuz< ._u_cmuee accum_mwciz< do acmucoo mv_cm;uuumocoz .H-H atem_a 43 ow pip wc:m_d EU m. 0.. : 523 o 400m 44 .mmooapm "apm .wmo_n_cmcwse— "m4 .mmowno__mo ”mu .A>\> .muflnuv om: "mumumom pzzum "Focmaocai: ;u_z quoPm>mc anamcmouwsoccu comma newncmommu >3 umuecmamm mew: mnceucmpm muewcaocggm new mmu_ce;oumm m:_u_=mmc esp .mhnxmfiv ewe—mo use cmm_:_mz cw emnWLUmmu mm mmmmmm .p.m .co an um__aa:mu mooxEouamcum sexy copueceamcq mme_=__mu a ;u_: coummmwu mm: pepcmuee accum_mmcuz< asp .; m cow mmoo:_mumuefiu cu_z covenao=_ coon we: was» mummpaouoea Eocd vm>_cmc .mcmsxpoa apneo_a_omcai_ocesum _eaou seed emceamca we: .e_cmume uceum_mmciz< .coFHmmmPn owe—appmo x5 Pe_cmpee pceum_mmciz< sage ummwmpmc mou_co:oomm .N-H at=m_e 45 e. EU mupmesmI a e a a e e.. 3 ea,“ 38 s .8. c :8. o d w iOON TV \\ A O 20 O 0 mo m4 7. .0m 46 the Streptomyces preparation (i.e. a B-glucosidase acting on cellobiose) and may arise from cellulose or possibly other glucans. AN-resistant material was subjected to methylation analyses (Fig. 1-3). Similar results were obtained whether samples were derived from protoplasts incubated with [14CJ-glucose for 1,3 or 21 hours. In most experiments, all of the radioactivity co-chromatographed with authentic 1,4,5 tri-O-acetyl-Z,3,6-tri-O-methyl glucitol, i.e., 4-linked glucose. Small amounts of radioactivity were detected in derivatives with higher retention times; these can be attributed to undermethylated products of cellulose. In some experiments small amounts of radioactivity (less than 15%), co-chromatographed with 3-linked glucose (Fig. 1-3). Thus, the labeled AN-resistant material is predominantly newly synthesized cellulose. Throughout the remaining text, the AN-resistant material will be referred to simply as cellulose. 3. Inhibition of cellulose synthesis by 2,6 dichlorobenzonitrile. DCB, at umolar concentrations, has been shown to specifically inhibit cellulose synthesis in cotton fibers and other systems (Montezinos and Delmer, 1980). DCB (3.3 pM) also inhibits incorporation of 14C-glucose into cellulose by soybean prot0plasts, by 67% as compared to controls lacking DCB (Table 1-2). Higher concentrations of DCB also inhibit incorporation of glucose into total 80% ethanol precipitable polymers. 4. The time course of cellulose synthesis. Experiments were undertaken to ascertain whether or not there is a lag period in cellulose synthesis during wall regeneration, as has been suggested by Burgess and coworkers (1978). Protoplasts were isolated from rapidly Figure 1-3. 47 Methylation analysis of AN-resistant material A. Gas liquid chromatogram of permethylated alditol acetates from a mixture of laminaribiose, cellobiose and cellulose. Peaks were identified by relative retention times. 8. Elution profile of radioactivity of permethylated derivatives from acetic-nitric resistant material obtained from protoplasts which had been incubated for 1 h. C. Same as 8, except the protoplasts were incubated for 3 h. 0. Same as 8, except the protoplasts were incubated for 21 h. Detector Response 48 Temperature (°C) Figure l-3 A 4-glucose i-glucose 4 a- glucose W PM us l65 ‘ l9)0 ' B r- W 1.... +- c- '" D —. ' "l 1 a I'll-'1 "5 "5 l65 |90 49 .Hoceguw do messHo> case do :ouuuuvm mzu Ha cmuecueLmu use .5 H to» assumcmaEmu soot ue use emucceo mew: mcouueazocH .mHoE:\_u: oH do Huu>uuoe uuuuomgm a guuz .25 H do eouuecucmucou Hague e m>um ou umuHaqzm we: mmoosz- ueHH .umueuuucu mcouuecucmocou ogu um emHHaazm.me3 moo .AHnH mHneu mmmv «can HH- ou ucmHm>us m Hmuucmuoa scum: Hague a ou oooe awe nuuz umucmEmHaasm .NHueu :5 m.o .eommz :5 m .m.m In .mmZImucu :5 oH cu umvcoamsmmc mum: HHE m cu moH x o.mv mummHQouoca NuH wHaeu 50 mm om Hm No .mume—eeuece coH x H Lee .5 H :_ emuegeeceecu emcee—m emcee—mimowHu meHeEee meueeHHemc sou meH > emece>ee meHeEem mueoHHemcm n m oHo.o ~.m m.m H m w oHo.o o.“ N. N m oHo.o «.0 w. H eHc.o ehHH Fir—4 mmo.o H.¢m MMN mom cum mmo eem mom «Km com Nmm ooH mam Nmm Nov mm mmm wmu mmv oH mmm mmm Hem m.m mwm mmm o coo.H mumegucxm emeHzHHee we ceuuueugcu a mceEHHee emeHeHHeo a euuec mmecw>e oemeHeHHee a omeHsHHee mcesaHea emece>e emeesz we :euumceaceecu ecu ce mu: we ueewum esp NiH m—neh a... mcestee HmeHHmece: :3 Heuou mmuou 51 growing, 2 day old liquid suspension cultures of soybean cells. The protoplasts were resuspended in a complex culture medium which contained sorbitol as an osmotic protectant (modified from that described by Constabel, 1975; see Materials and Methods). The total water potential of the medium was approximately -19 bars. The final concentration of glucose in the medium was adjusted to try to provide: (i) an adequate carbon source to sustain development for the duration of each experiment and (ii) a specific activity of the supplied label sufficiently high to permit detection of newly synthesized polysaccharides after the tracer is diluted by the internal glucose pools in the prot0plasts (as is indicated in the legends for Figs. 1-4, 5 and 6). Under these conditions, 14C-glucose is incorporated into cellulose within 15 min and the amount of incorporation increases over 180 min (Fig. 1-4A). There is a concomitant increase in the 14C content of total 80% ethanol-precipitable polymers (Fig. 1-48). [14C]-Glucose incorporation into cellulose continues to increase over 12 and 24 h (Fig. 1-5 and 6). The rate of incorporation increases in the 12 h time course and is roughly linear in the 24 h time course. These differences may reflect varying rates at which 14C-glucose, at different supplied concentrations, accumulates in metabolic pools of the protoplast until it approaches a constant specific activity. These studies provide chemical evidence that cellulose is synthesized without an appreciable lag by soybean protoplasts during wall regeneration." 5. Deposition of fibrils on the surface of the plasma membrane. ProtOplasts were fixed in glutaraldehyde immediately following the isolation procedure. Transmission electron microscopy of the protoplast indicates that the surface of the plasma membrane is devoid 52 .meumewHeae we emece>e mgu mu ucwee comm .Hza mw.o :ewuecucmecee emeesHm Hmcww .eHeE:\wu: m.~v emeeeHmimszu Hu cuwz eeuceEeHeezm seweee :ewuececemec HHez cw eeueowecw mmewu Lew emceuHeo ecu: NHE m cw moH x av mumeHeeuecq .mcestea ewmeHaHHmecec cw see .m emeHzHHee cu Ego .< .sceu ucegm ”mceexHee «HemuweweeceuHecegue ecu emeHeHHee eucw mmeoszumueHu we :ewueceeceecH .e-H aeemwu 53 2:5 as: 2:5 95% cm. ON. 0m 0 Om_ Om - OOON OOOd O 019 000m - ooom flogged e_me_:__eecez .m 30.260 .< e-H at=e_a 00. undo 54 .meueewHeee we emece>e ezu mucmmecaeg uewee seam .Hze m.w eewuaeueaeeee emcee—m Haeww .a_eEa\_ea m~.ov ameee_e-HH=Ve HH eu_z eeucmEmHaqsm sawees :ewuecmcmmec HHez cw emueowecw meswu ezu Lew emcauwso mew: HHS m :_ ooH x mm.Hv mummHeeuece .mceEzHee ewmeHaHHeoce: cw Ego .m emeweHHee :w =ae .< .emceee eewu .; NH "mceezHee segue ecu ewe—:HHee eucw mmeeaHmimueHU we :ewueceeceecH .m-H aeeewd 55 X Ar: 2.2:. 93.53..— sm.©.00¢m0 N_0_009N00 .. 000.0. x . 000.. D 3 d d . 08.er .. eeed w . 000.0m - 000.m mceEzed 28.20082 .0 eme_:__e0 .4. Figure 1-6. 56 Incorporation of [14CJ-glucose into cellulose: 24 h time course. Protoplasts (4.6 x 106 in 5 ml) were cultured for the times indicated in wall regeneration medium supplemented with [14C(U)]-glucose to give final concentration of 20 mM, at a specific activity of 0.1uCi/umole. 2000- pm 0 IOOO- 57 HOURS Figure l-6 58 Figure 1-7. Time dependent deposition of fibrils on the surface of protoplasts. A. The surface of a protoplast fixed immediately after isolation (x28,000). B. The surface of a protoplast that was cultured for one hour (x31,000). C. Fibrils present on the surface of protoplasts that had been cultured for 3 h (x50,000). 59 Figure l-7 60 of wall material (Fig. 1—7A). Protoplasts fixed after one and three h in culture show numerous microfibrils on the surface of the plasma membrane (Figs. 1-78 and 7C). The distribution of fibrils on the surface of protoplasts is not uniform, possibly reflecting some loss of fibrils during fixation. These observations reinforce the results of the time course experiments (Section 84), i.e., that there is no lag in cellulose synthesis during wall regeneration. The absence of pre-existing fibrils on the protoplast surface (see Fig. 1-7A) indicates that the 14C-glucose is incorporated into newly synthesized 4-3-glucan chains. C. Synthesis of Other Cell Wall Components For reasons discussed in the Introduction it is difficult to identify and isolate the cell wall during the initial stages of wall regeneration. Therefore 14C-glucose labeled protoplasts were divided into two fractions, by collecting intact protoplasts (including that portion of the regenerating wall which adhered to the plasma membrane) and then isolating total ethanol-precipitable material from the spent protoplast culture medium (ECM). These fractions were utilized to examine the synthesis of non-cellulosic wall components during the first three of wall regeneration. For comparison, cell walls were prepared from one and two day old cultures of soybean cells grown in liquid suspension culture. Ethanol was added to the spent culture medium to precipitate polysaccharides and glycoproteins (secreted polymers). 1. Neutral sugars. The neutral sugar composition of soybean cells walls and extracellular polymers is presented in Table 1-3A. 61 This analysis gives the composite neutral sugar profile for all of the polysaccharides and glycoproteins in the fraction, except that of cellulose. The distribution of sugars is different between the two fractions: Rhamnose is present only in the cell wall, glucose accounts for the largest proportion of the neutral sugars in the wall. Xylose, followed by galactose are the predominant sugars of extracellular polymers. Protoplasts were incubated in the presence of 14C-glucose for 3 h and total 80% ethanol-precipitable polymers were isolated from the intact protoplasts and the spent culture medium. The distribution of label in the monosaccharide constituents of these polymers was determined by GLC as described in the Materials and Methods (Table 1-38). During the 3 h of culture, 14C-glucose is converted to various nucleotide pentoses and hexoses; these serve as substrates for the synthesis of polysaccharides and/or glyc0proteins (the 80% ethanol-precipitable material). The distribution of label in the protoplast fraction is distinct from that of the ECM: arabinose, mannose, and glucose are the major components of the prot0plast fraction, with smaller amounts of galactose, xylose, and rhamnose + fucose. Arabinose, galactose and glucose are the major components of the ECM, with smaller amounts of mannose, xylose and rhamnose + fucose. The distribution of 14C label in newly-synthesized polymers in both the protoplast and ECM fractions also differs from the overall composition of the cell walls and excreted polymers of liquid suspension cells (c.f. Table 1-3A and 3B). These experiments demonstrate that protoplasts synthesize a variety of glycoconjugates during the first three h of wall regeneration. 62 .mceozm maewce> ceezuee wge>eeee cw meececewwwe Lew cewueeccee ceuwe .cewuwmeeeee a eHeE emece>e ecu me eemmecexe ecu meHQEem eueewHeae co eeacewcee we: xceecmeuesecco .meezues use mHewceuez egu cw eeewcemee we eeceaeca ewe; meHeEem .eHeE:\wuncH we auw>wuoe ewwweeem e e>wm eu 25 H we :ewuecuceecee Hecww e um emeesziueH sup: .; m Lew eeueeeecw ewe: mumeHaeueca .mumeHaeuece Eecw ee>wcee meewcegeeemxHea eermegucxmutze: we :ewuwmqueu semen Haguee: es» .waaH .ceEHeo ecu ucecwez cw eenwcemee me .meczuxwa Leona eceeceum we me>wue>ucee :e eemeev cemzm some cow ng>eeec cw meocecewwwe Lew cewuoeccee ceuwe .cewuwmeeeee ereE eemmecexe ewe meeHe> .mecauxwa cemem eceeceum we me>wue>wcee we meewu :ewuceuec esu eu :emwceQEee an eewwwuceew ewe: meueueoe HeuweHe egw .meesuez ecu mHewceuez egu cw eeewcemee me ee:_Eceuee ewe; meewcezeeemaHea eeueceem use mHHez HHee esu we mcewuwmeasee cemem Hecuzez mum wueeeweec we cewuaewcumwo .m mm KN 0N 0H m e HN 0N NH mH m e m m Nae N Nee H mHHez HHee emcee—m emeueeHem emeccee ewe—ax emecweece emeuzw eme:Ee;c .Aa eHeEv meweuHee HHee :eeexem Eeew mceEHHea eeueweem nee mHHe3 HHee wo .< mcewuwmeeEee Leann Hagueez niH epnew 64 2. Methylation analysis. GLC of permethylated alditol acetates from cell walls of two day old liquid-suspension cells and the secreted polymers from the Spent medium illustrate the spectrum of sugar linkages present (Figs. 1-8A and 1-88). These derivatives are typical of the cell wall and secreted polymers synthesized by dicots. For example, i) Terminal-arabinosyl and 3,6-linked galactosyl residues are characteristic of soluble arabinogalactan hydroxyproline-rich proteins (Lamport, 1977; Clarke 33 al., 1979). These are predominant in the secreted polymers. ii) 4-linked glycosyl residues characteristic of cellulose and starch are prominent in the profile of the derivatized cell walls. iii) 4-6 linked glucosyl residues are characteristic of hemicellulosic polysaccharides. Uronic acid residues, characteristic of pectic polysaccharides are not identifed by this type of analysis. Labeled polysaccharide-containing polymers from protoplasts and the ECM were subjected to similar methylation analyses as described in the Materials and Methods. Due to lack of suitable standards, these data have not been corrected for differences in recovery between individual derivatives. The elution profile of radioactivity indicates that a variety of pentosyl and hexosyl residues, i.e. sugar linkages,- are present in the newly-synthesized polymers (Figs. 1-88 and 80). These include: i) t-Arabinosyl and 3,6-linked galactosyl residues in the polymers derived from protoplasts may represent the synthesis of arabinogalactan proteins, destined for secretion into the extracellular medium. These residues are heavily labeled in the ECM fraction. Alternatively, all or part of the t-arabinosyl in the protoplast fraction may represent araban, a pectic polysaccharide commonly found in dicot cell walls. ii) t-Xylosyl and 4,6- linked glucosyl residues 65 .ecsuHee umuHeeuece we c m ceuwu seweee cewuucecemec HHuz Eecw eeuuHemw sow ecu Eecw eecueece me>wuu>wcee cw Huw>wueueweuc we ewaece cewquu .o .< cw eEum ecu ecu mcewuuewwwuceew cued .meecuez ecu mHuwceuuz cw eecucemee mu eeceucmeuusecce ecu eeruu>wcee .eeuuHemw mu: Huwceuue ecu .mcewmceemem HHeo we sewees eceuHee ecu eucw eeueceem mceExHee we mesuuu>wcee eeuuchueEcee we ewaece cewuaHe 040 .0 .muceawceexe cuHuEwm Huce>em we Huengu mu ewaece ecu .mecuecuum eeHecuHc: cuwz cewqueuee Hc eewwwuceeu ecez mcewueucw ecu .meHesum euuewHeee ce eeEcewcee mu: Hceucmequecco .eeuuHacueE ecu cewuumewwcucee ac eeueeHHee ecez mumuHaeuecc .HzE H cewuucuceecee emeeeHm Hucww meHeE:\wu: oHV emceeHm -mHevuvHu cuwz eeuceeepeeem seweee cewuucecemec HHuz cw c m cew eeceuuze ecez .HHE m cw mcH x o.mv mumuueeuecc .cewueucw umuHeeuece eHecz ecu eecw eecwuuce me>wuu>wcee eeuuuxcueacee we xuw>wueueweuc we ewaece cewuapm .m .Aecuecuum Hucceucwv HeuwmecwnNH HameueuHumim.muHH “ce~wewumuHe + meeesumum.enoH meeueuHumum.eum “meeueuHumuonm "meexecueuw "HameeeHmnmuo "HameHxxie ecu iNum “HameueuHumiuue ”Hamecwcucuum.Hnm “HameHzxiuNN "meecwcucuuunH "mezewmec meeexuu we eauccwH eeeeeee ecu eu cewec mcece:c cued .checm uec uuuev mwmxwucu Hucueeem mmue ce\ecu .mecuecuum cuwz cemwcuaaee .meswu cewuceuec e>wuuHec Nu eewwwuceew ecez mcuec .meecuez ecu mHuuceuuz cw eecwcemee mu eeNHuu>wcee ecu mHHee eceuHee coumceemem eHe Hue N Eecw eeuuHemw ecez mHHu; HHeu .mHHuz HHee cuecaem we me>wuu>wcee eeuuchueEcee we ewaecq cewque ecu .< meewcuceeumaHea cuecxem we memxuucu cewuuHchez .u-H aesmwu 66 00. Gav ecu—eceeEeh 00. 0v. 0.. em. =me. e: m: mnH eczmww . Gav eceueceacce... 9. cu. me. 4 9. l 2 00¢ . on M “i .00— 43 e .Hunxw nxumHMw = e c nxum” 00m. H e . 00¢ 0.2000 9. em. me. e: m: N. asuodsaa JOIOGISQ asuodseg JOIOGIGG 3 Au mm 67 in protoplast polymers may represent xyloglucan, a hemicellulosic polymer found in dicot cell walls (Albersheim, 1976; Darvill gt al., 1980). iii) t-galactosyl, 4- and 6-linked Galactosyl residues may also indicate the presence of galactans (Darvill 33 al., 1980). The cumulative proportion of radioactivity in each type of gly- cosyl residue (i.e., t-glucosyl plus 4-linked and 4,6-linked glycosyl residues) is generally consistent with the neutral sugar analyses (Section C-1). Again, the labeling patterns of polymers in the proto- plast and ECM fraction are distinct and differ from the composition of the cell walls and secreted polymers. These data are insufficient to permit definitive identification of most cell wall and extracellular polysaccharides synthesized by protoplasts during the initial stges of wall regeneration: this requires separation of individual polymers and subsequent detailed analyses for each. Nonetheless, these results indicate that a wide range of complex polymers, including pectins and hemicelluloses, are synthesized during the first three hours of wall regeneration and these bear some Similarities to the cell wall constituents and secreted polymers from liquid-suspension cells. 3. Synthesis of 3-3-glucan. No appreciable quantity of 3-linked glucan is detected by methylation analysis in the cell walls or excreted polymers of liquid-suspension soybean cells (Figs. 1-8A and 1-88). However, protoplasts, fed 14C-glucose, synthesize significant amounts of 3-linked glucan (Figs. 1-88 and 80). This is most probably 3-8-glucan, a polymer whose synthesis is induced by wounding and which is also present in Specialized tissues: e.g., pollen tubes, and cotton fibers (see Chapter 3, Darvill gt al., 1980). 68 Discussion The Early Events in Cell Wall Regeneration. Soybean protoplasts were cultured in the presence of 14C-glucose to examine the synthesis of polysaccharides and glycoproteins during the initial stages of wall regeneration. ,The relative amount of glucose incorporated into polymeric material is influenced by the solute concentration and composition of the culture medium (Table 1-1). Cell elongation in whole plants is slowed by.a drop in leaf water potential from -2 bars to -12 bars (for a review see Hsiao 1973). However it is difficult to acess whether these represent the same phenomenon, that is an inhibition of the synthesis of cell wall material, because protoplasts require a culture medium with a maximum water potential of at least -12 bars. Cellulose Synthesis. Microfibril deposition on the surface of the plasma membrane is characteristic of the early stages of wall regeneration (Willison, 1976). In most previous studies these fibrils have been equated with crystalline cellulose, without rigorous characterization. Protoplasts, isolated from rapidly growing suspension cultures of soybean cells, were incubated with 14C-glucose and this label was incorporated into ethanol-precipitable polymers. A very small proportion ((5%) of these newly-synthesized polymers are resistant to hydrolysis with AN reagent; this labeled AN-resistant material is predominantly newly-synthesized cellulose (Figs. 1-1, 2 and 3). Additional experiments demonstrate that soybean protoplasts begin to synthesize cellulose within 15 min after the protoplasts are transferred to culture medium (Fig. 1-4). 69 Cytological observations indicate that the surface of protoplasts, fixed in glutaraldehyde imnediately after isolation, is devoid of microfibrils; after one or three hours of culture, numerous fibrils are visible (Figs. 1-7). The initial absence of microfibrils suggests that the labeled glucose is incorporated into new cellulose chains and not preexisting microfibrils. These findings indicate that the cellulose synthesizing machinery retains its capacity to synthesize cellulose during the isolation of protoplasts from these rapidly growing soybean cells. In contrast, Burgess and coworkers (1978) observed a "lag" period of eight h before protoplasts derived from leaf tissue of Nicotiana begin to synthesize microfibrils. They suggested that this "lag" period is a comon feature of cell wall regeneration. It is more probable that this lag period is a function of cell type and perhaps cultural conditions: Protoplasts from slowly growing tissue or mesophyll cells may have to synthesize the cellulose synthetase 9g ngxg, prior to microfibril deposition. Low water potential and high salt concentration also inhibit wall regeneration (Herth and Meyer, 1978; Willison and Klein, 1981). Other cell wall components. There have been a limited number of studies which partially characterized, at a biochemical level, the noncellulosic components of the regenerating cell wall (see Willison and Klein, 1981). Hanke and Northcote (1974) reported that during the initial 20 h of wall regeneration, soybean protoplasts utilized exo- genous glucose primarily for the synthesis of starch, protein and cellulose. They suggested that little other wall material is synthe- sized during the first day of wall regeneration. In contrast, Asamizu 70 and Nishi (1980) reported that over the same time scale, carrot protoplasts synthesize pectins, hemicelluloses and cellulose. In this study, soybean protoplasts, incubated for three h with tracer levels of glucose synthesize a variety of polysaccharide containing polymers (Table 38, Figs. 88 and 8D). The newly-synthesized polymers contain a variety of glycosyl residues (Table 1-38 and Figs. 1-88 and 80). Of the neutral sugar residues in these polymers (excluding cellulose) only 30% of label in the protoplast fraction is found in glucosyl residues; the distribution of glucosyl residues in these polymers suggest that starch is not a major component of these newly-synthesized polymers. This non-cellulosic glucose may be derived from 3-3-glucan, xyloglucan and other cell wall polysaccharides. Alternatively, part of the glucosyl residues may represent cytoplasmic polysaccharides or polysaccharides synthesized within the cell but destined for secretion to the wall or culture medium. Substantial amounts of mannose in these samples represent a distinct difference in the composition of this soybean wall material as compared to that of the primary cell wall of other cultured cells which have been studied in detail (Darvill _t._l., 1980). Other t-pentosyl and glycosyl residues in the newly synthesized polysaccharides are characteristic of a variety of pectic and hemicellulosic polysaccharides and glycoproteins similar to those of the wall and secreted polymers of liquid-suspension cells. Additional work would be required to identify which of these newly synthesized polymers form the regenerating wall or how they are assembled and how their synthesis is regulated as compared to that of the cell wall 71 of suspension grown cells. In contrast Significant amounts of 3-B-glucan are synthesized by protoplasts during wall regeneration (Figs. 1-88 and 80). Little or no 3-5-glucan is present in the secreted material or cell walls of suspension grown cells. This synthesis of 3-B-glucan may represent a wound phenomenbn or may be part of a transient phase of wall regeneration. The 3-B-glucan synthetase is believed to be a marker enzyme for the plasma membrane (Leonard and Hodges, 1980;'Quail, 1979). In many tissues the activity of the enzyme is enhanced by wounding (Brett, 1978) or environmental stress (Smith and McCully, 1977); in other instances the enzyme is under developmental control (Maltby gt al., 1979). The glucan-synthesizing enzymes of soybean cells are examined in the following chapters. Attention is focused mainly on the 3-B-glucan synthetase because of its importance in cell wall regeneration. In addition, the information accumulated about this enzyme suggests its isolation and characterization may provide a good starting point for understanding the enzymology of cell wall biosynthesis. CHAPTER 2. SOLUBILIZATION OF B-GLUCAN SYNTHETASES Results The 3-B-glucan synthetase and cellulose synthetase are believed to be components of the plasma membrane (Leonard and Hodges, 1980; Quail, 1979). The synthesis of other non-cellulosic cell wall polysaccharides is believed to be associated with endomembrane systems (Ray, 3 al., 1976). Thus far, none of these enzymes has been purified or fully characterized. As an initial step towards the isolation of the 3-a-glucan synthetase, experiments were undertaken to prepare a solubilized form of the enzyme and to define conditions to maintain enzyme activity for subsequent purification procedures. A. Influence of pH and Sorbitol Concentration on Glucan Synthetase Activity in Crude Membrane Preparations. When e-glucan synthesis, from UDP-glucose, is assayed in a buffer- ed medium (pH 5.5) lacking an osmotic stabilizer, only very low amounts of product are detected (Fig. 2-1). Glucan synthesis is enhanced by including 400 mM sorbitol in the assay buffer at pH 5.5. If the pH is raised to 7-2 (by substituting K'TES for K-MES), in the presence of sorbitol, there is a further substantial increase in enzyme activity. Unless otherwise stated, subsequent assays for B-glucan synthetase activity utilized buffers at pH 7.2 and contained 400mM sorbitol. 72 Figure 2-1. 73 Effect of pH and sorbitol concentration on glucan synthetase activity from a crude membrane fraction. 11 g of 2 day old cells were disrupted in 50 mM K-TES pH 7.2, 1 mM DTT, 10 mM MgCl2; 400 mM sorbitol. The brei was filtered through two layers of nitex cloth (40 um, average pore size) and the filtrate was centrifuged at 1,000 x g for 5 min to remove cell wall fragments. The supernatant was divided into 4 aliquots, these were centrifuged at 25,000 x g for 30 min. The resulting pellets (crude membrane preparation) were resuspended in 2 ml of one of the follow buffers: i) 50 mM K~MES, pH 5.5, 1 mM DTT, 5 mM MgCl ; ii) same as (i) + 400 mM. sorbitol; iii) 50 mM K-T S, pH 7.2, 1 mM DTT, 5 mM MgCLg; or iv) same as (iii) + 400 mM sorbitol. Each reaction contained approximated 92 ug of protein. UDP-gluCose was supplied to give a final concentration of 0.1 mM, with a specific activity of 5 uCi/umole and the reactions were incubated at 25°C. At intervals indicated, duplicate aliquots of 0.2 ml were removed and pipeted directly into 0.8 ml of 100% ethanol. Total glucan synthesis was determined as described in the Material and Methods section. Total radioactivity in individual samples ranged from 1,500 to 55,000 cpm. nmoles glucose incorporated mg protein 700 600 74 pH 7.2 . +sorbiiol 0 ‘:pH 7.2 4; pH 5.5 +sorbitol #3- pH 5.5 60 ' minutes Figure 2-l 75 These crude membrane fractions probably contain plasma membrane and some endomembrane vesicles, both of which are likely sites of glucan synthesis (Leonard and Hodges, 1980; Ray, 1980; Quail, 1979). Thus, although the assay conditions would probably have favored a plasma membane localized 3-3-glucan synthetase (glucan synthetase II; high UDP-glucose), the measured glucan synthetase activity may represent the products of more than one enzyme (Quail, 1979). B.“ Detergent Solubilization of Glucan Synthetases Several detergents were screened for their effect on enzyme activity and their potential to solubilize the glucan synthetases. These included lipids, neutral detergents and one ionic detergent, cholate (Fig. 2-2). A crude membrane pellet was resuspended and stirred in buffer plus detergent; each detergent critical micelle concentration, and/or two to five times its critical micelle concentration. Ater 15 min. an aliquot was removed to measure total glucan synthetase activity (first 30,000 x g pellet). Half of the remaining sample was centrifuged at 30,000 x g and enzyme activity was measured in the resulting supernatant (solubilized fraction) and pellet (second 30,000 x g pellet, which was resuspended in assay buffer without detergent). The remainder of the sample was treated with detergent for an additional 130 min. and then centrifuged as described above. Phosphatidyl choline, noniondet P-40 and octyl-B-glucoside treatments do not solubilize appreciable amounts of glucan synthetase (Fig. 2-2). Phosphatidyl choline and noniondet P-40 both slightly 76 .eeuuewecu mu cewueucw ceue cew Huw>wueu Huueu emuce>u ecw .EQe coo.mo eu oom.H c.ecw eeccuc muesee.a eeHecuH ecu cw Huw>wueueweum .cwe om cew eeuuccecw ecez mcewueuec “HeHesn\w0: m uuv emcee—mica: :5 H.o cuwz .cewueucw ceue we mueeewHu HE N.o cw .euuewHaae cw eeHummu mu: Huw>wueu eEHNcu .ucemceuee ueecuwz cewwec cw eeeceemcmec mu: ueHHee ecuccses eceeem ecu .cewuumewwcucee ecu ucesuuecu ucemceuee ceuw< .cewwwcem eeHu cuuc u cw eeuuewcem HHwewcc mu: eHeEum ceue Hewemww ecu cw eeuuewecw mcewuucuceeceo ecu uu eeeeu mu: ucemceuec .meHeEum HE N .ucmwe euew eaeusuu eea HHE tea eueueta we we um.e uec .Heuwetem 2e ooe .mueez as m .uue 25 H .N.w :a .muw.x :5 cm .cewwzc we .5 0H cw eeeceememec mu: cewuucuaece ecucceee eeece ecw .H-N ecsmww cw eecwcemee mu eeueecmwe ecez mHHee eHe Hue e ecu N we a HN .mcewuucueece ecuccees cw cewuzcwcumwe ecu Huw>uueu emuuecuch cueeHm ce mucemceuee we eeceechH .~-~ aesmuu l5 min 77 s? s I '2 g g. 0 IZ////// a _ E lZ/////[ 2 g .3; 3. "‘ 3. 3 a? 8 g S 6.6 3 W////// I E E E rz///// 5 E w 3 g 8' W ‘ Ed D E////// g a— E I g g IQ////////////// , 1517/////////// f U 2' 2 _ " 5 E///////////////// [/1////////// v8 2 '3 E 8 O a if E Z 2 1}, 3 W//////// [’[f///////// 23 N O '0' 2 m 5 O I 1 . d) (o ,5. pemodaooui esoanib eeiouiu mo; Figure 2-2 G 78 inhibit glucan synthesis (Fig. 2-2); Tween-20, digitonin and Triton X 100, other neutral detergents, also slightly inhibit enzyme activity (data not shown). This inhibition of glucan synthesis may result from disruption of a favored conformational state of the enzyme within the membrane (Helenius and Simons, 1975). Octyl-B-glucoside severely inhibits glucan synthesis in the first 30,000 x g pellet fraction; the a-glucosyl_moiety may act as a competitive inhibitor as it bears some similarity to the substrate UDP-glucose. However when some of the detergent has been removed by centrifugation (e.g. after the initial treatment with 20 mM octyl e-glucoside) the second pellet fractions (assayed in buffer without detergent) exhibit at least as much glucan synthetase activity as the control samples (Fig. 2-2). Glucan synthesis is inhibited in all fractions after treatment with 40mM octyl-a-glucoside. (Na) cholate, an ionic detergent, at pH 7.2 stimulates glucan synthesis (Fig. 2-2). This may represent increased substrate accessibility or altered enzyme conformation (Helemius and Simons 1975). Cholate (ISmM) treatment solubilizes a small proportion of the total enzyme activity in 15 min. Solubilization is enhanced when the concentration of cholate is raised to 30mM and the detergent treatment is extended to 145 min; approximately 50% of the total glucan synthetase activity is solubilized. By raising the pH of the buffer to 7.8 (the pK of cholic acid), approximately 80% of the glucan synthetase activity is solubilized after 15 min. of treatment with 30 mM K cholate (Fig. 2-3). (In this experiment, the second pellet fractions were assayed in the presence of (K) cholate). To Optimize conditions for solubilization, the ionic 79 .eeueucucsm mu: Heee omv eceecmceuc ceuwu Eco coo.HN eu coo.H Eecw eemcuc mueeeece echuwaweecauHecucue eeHecuH ecu cw Huw>wueueweuc .mcwe ON cew eeuucaecw ece: mcewueuec ”HeHeEn\wu: oHV .zs H.o we cewuucuceecee Hucww u uu eewHeeem mu: emeesHaueoe .cewueucw ceue we mueeewHu HE N.o cw eecHEceuee mu: Huw>wueu eEHNcm .uHum cuw: cewwec cw eeeceemsmec mu: ueHHee mwcu cue—Hen ecucceee eceeem u ecu mcewueucw eHceHem eucw eeuucueem ece: meHeEum ecu ceuw< .eeuuuwecw mcewuucuceecee ecu uu ceue eu eeeeu mu: Hux ecu meHQEum e eucw eeew>we mu: cewmceemem ecuccses ecw .NHumz 25 m .euuHece.x :5 on .wwc :5 H .w.w :e .muw.x 25 cm mcwcwuuceo cewwcc we .5 w cw eeeceemamec mu: cewuucueeca ecuccsee eeece ecw .HuN eczmww cw eecwcemee mu eeuezcmwe ewe: mHHee cuecHem eHe Hue m we a a ocopuwNm—wnz—Om vmuwwtmElmumposu :0 cowHMLucmocou upwm $0 muumwh—m .m-~ atemue IOOmM KCI I5 min no additions ZOOmM KCI 50 mM KCI 80 E first Pellet 8 second Pellet CI second Supernatant lei—l en Nibfi‘ ., “6' ‘ \. (“VACXZ’K l”'\t\’/ . ’I _. iv . ' 3 . \ $834492» l .“‘=.\' eye/.923 ‘- 4:570, -,'-: ~ ' i W, ,, .. ,.,« ,4 ,, .7 . A .. . a $3. "#33. 3,5,, §th43 J, . , _. .. \n ._\- . \;:.\m-;*.)z-\._, s. ?;,.m4.~,-v.4r _. . _ =r , , .. ‘ Rifle": ' 1‘ .‘ "a . ”st-x"). .- 1- 2-4-m\&24«més%t.%} I . - . 6 IZO min 1 N peiolodloaug esoanib selomu logo; L Figure 2-3 (I 81 strength of the buffer was increased by varying the concentration of added KCl. However high salt concentrations do not enhance solubilization (Fig. 2-3) and final concentrations of KCl at.z 50 mM actually inhibit glucan synthesis. Removal of cholate from the soluble enzyme preparation, on a desalting column, results in loss of glucan synthetase activity and precipitation of the protein (data not shown). The solubilized enzymes contain at least two glucan synthetase activities (these results are described at the end of section C). These experiments define conditions for preparing solubilized forms of membrane-associated glucan synthetases. Of the detergents screened only cholate solubilizes glucan synthetase activity. The solubilized glucan synthetase(s) is probably part of a micellar complex consisting of enzyme, cholate and, possibly lipid (Helenius and Simons, 1975). Removal of cholate from the solubilized enzyme preparation, by gel filtration chromatography (on Bio-gel P-6), resulted in the precipitation of protein and loss of glucan synthetase activity (data not shown). Pure cholate micelles, in contrast to many other detergents, are relatively small (MN 2,200 daltons; Helenius and Simons, 1975). Therefore the molecular weight of the mixed enzyme-cholate micelle would be expected to be similar to that of its constituent protein and thus within a range amenable to gel filtration chromatography even in the presence of detergent. C. Preparation of Solubilized Glucan Synthetases. 1. General protocal. Paramethylsulfonylfluoride, (PMSF, lmM) was routinely added to all buffers, immediately prior to use, to Figure 2-4. 82 General protocol for preparing solubilized glucan synthetases. See Materials and Methods Figure 2-4. collect on Miracloth Plasmolyze Disrupt Remove wall fragments Detergent treatment: incubate with stirring for 15 min at 4°C 83 two day old cultures wash cold H20 wash cells Resuspend In 400 mM sorbitol 50 mM K-TES pH 7.2 DTT PMSF MgClz iii l Pass thru a Ribi press at 8000 PSI, at 5-10°C | filter thru Nitex cloth brei I 500 x g, 5 min wall debri 30,000 x g, 5 min. l first membrane soluble enzymes, pellet small organelles Resuspend in 400 mM Sorbitol 50 mM K-TES pH 7.8 30 m1 BTP-cholate 1 mM DTT 1 mM PMSF 5 mM MgCl2 Determine enzyme activity [30,000 x g, 45 min. I ’I second membrane soluble pellet glucan synthetase preparation 84 . .an ooo.oo cu com Eocw woman; muoavoca cowuommc mzu cw xuw>wpomowumm .cws cm cow umuuaaucw mew: meowuucmm .Acowumcucmocou Pmcww AA>\>VV gm.w~v pocooxpa wo cowuwucm as» cmuwm umcammoe ompm we: xuw>wuom msx~cm .mpoaz No.o\wu: ~.o wo zuw>wuum uwwwumam a m>wa o» :5 H.o wo cowpmcacmucou —m:ww a an umwpaazm mm: mmouz—muaos mxuw>wuom mmwumgucxm coon—m cow .maaow—azu cw .umxammm we: :owuumcw comm .Amuo—ogu ANV van cwmuoca .uwnwpv me mmm umusawmz amp—ma acmcaema acoumm as» .ucmsummcp acmmcmumu cmuw< .ms cam umzmwoz :owumccamca meccasms ounce mzw .cmwwan cw umaazcmwu ago: mppmo camaxom vpo xmu N we .m m.w~ HnN m—nch 85 emza :5 H .wwa :2 H .wae_o=o.¢wm :2 om ._ou_ac0m as see .eommz :5 m .m.~ Ia .muw-x :5 om cw 3.. wmza :5 H .who 25m .—ouwngom :5 oce .eommz :5 m .~.m In .muwux :5 om cw we oe.mm mm.m m~.~ oHo.H N.c~ oH.o npcaumcemazm xom new om.m «.mfi oe.o CNN mas mmw mm.e nua__mg gem new «.mH o.m~ -.~ ooe.m HNN ¢¢.o a»m__ma xom end ©H.e cod. ems. mmN m.Ho we“ “NH auceuaczmaam xom um“ c“.~ mos. New. N.m~ o.¢m - m~.m ecu—.ma mxoom mm.“ mom. mme. cfic.~ ccH.H “NH emueemmoso; _auow News om cmnwl awe umuacoacoocw Away Pocmox_mu\+ can as can mm—oE: mmouzpm mmposc usmwwz Away owumc Pocmux_m+ xuw>wuum uwwwumqm wocmo>9m+ xuw>waum punch amp—ma :wwuoca page» cowuumcw mommumgucam cmuapm m.a:.om wo cowuccaamca film m—nch 86 protect against serine proteases. PMSF did not inhibit s-glucan synthesis (data not shown). This suggests that serine residues are not involved in the active site(s) of this glucan synthetase. The general protocol for preparing solubilized glucan synthetase(s) is illustrated in Fig. 2-4 and described in more detail in the Material and Methods Section. Table 2-1 summarizes the distribution of glucan synthetase activity in the various soluble and membrane pellet fractions. The proportion of the total protein from the first membrane pellet, which was solubilized by cholate, varied considerably (25 to 90%). Solubilization is enhanced if the ratio of cholate buffer to initial pellet weight is at least two ml per 100 mg. This illustrates that solubilization is not only a function of detergent concentration pg; gg but is also dependent on the ratio of detergent to membrane (Helenius and Simons, 1975). It should be noted that the total amount of enzyme activity in each fraction may be altered by a number of conditions which are varied between successive fractionation steps (Table 2-1). These include: 1) pH: Glucan synthetase activity was assayed at pH 7.2, for the total homogenate, 500 x g pellet and first 30,000 x g supernatant (soluble enzymes and small organelles). For the remaining fractions, glucan synthetase activity was determined at pH 7.8. 2) Cholate: Glucan synthetase activity was measured in the presence of 30 mM cholate in the first and second 30,000 x g pellet and the second 30,000 x g supernatant. Cholate stimulates glucan synthetase activity as compared to control samples without detergent (Fig. 2-2). 87 Only 12% of the glucan synthetase activity from the first 30,000 x g membrane pellet was recovered in the solubilized enzyme fraction (c.f. total activity: 221 nmoles glucose incorporated versus 26 nmoles incorporated). Four times as much glucan synthetase activity was found in the second membrane pellet (113 nmoles incorporated). This distribution of glucan synthetase activity contrasts with results obtained in previous experiments (Figs. 2-2 and 2-3). However, a possible explanation for this discrepancy is that the glucan synthetase activity is not proportional to protein concentration under these experimental conditions (as was observed in a preliminary experiment). In the experiment summarized in Figs. 2-2 and 2-3, the solubilized enzymes were assayed directly (at approx. 580 ug protein per ml). In the experiment summarized in Table 2-1, total glucan synthetase activity was determined from an aliquot of the solubilized enzyme fraction, which had been diluted (final protein concentration 94 ug protein per ml). The amount of membrane lipid present in each fraction under these assay conditions also varied. Davies (1977) has speculated that lipid concentration may influence the activity of membrane- associated aspartate kinase measured in vitro. The addition of glycerol to the solubilized enzyme preparation results in a substantial stimulation of glucan synthesis (40 to 100 fold). Glycerol activation of a particulate glucan synthetase, derived from Lupinus albus, has been reported by Thomas and Stanley (1968). Enzyme activity, measured in the presence of glycerol, (16.5%, v/v, final concentration), is stimulated in all of the fractions (Table 2-2). The ratio of stimulation (+/- glycerol) generally increases during the fractionation and is highest for the solubilized glucan 88 synthetases (38 fold). The distribution of glucan synthetase activity between the second 30,000 x g pellet and the soluble fraction is now reversed: The soluble fraction has four times as much total activity as the pellet when both are assayed in the presence of glycerol. (Total activity in the soluble fraction: 1,010 nmoles glucose incorporated versus 270 nmoles in the pellet fraction.) The solubilized enzyme fraction was stored with glycerol (final concentration 33%), then diluted 1:1 with cholate buffer prior to use. Enzyme activity is stable for at least four weeks at temperatures of -40°C and below. Enzyme activity is also stable in the presence of glycerol for at least 24 h at 4°C. In an effort to protect enzyme activity during subsequent purification, glycerol (final concentration 16.5%) was included in the cholate-based buffers. 2. Product analysis. The ethanol-precipitable in material synthesized by each fraction (from Table 2-1) was subjected to methylation analysis and the distribution of radioactivity in the products was examined. Table 2-2 details some preliminary results. In the products of the solubilized enzyme preparation more than 60% of the radioactivity is found in 3-linked glucosyl residues. The remainder is found primarily in 4-linked glucosyl residues and some t-glucosyl residues. Radioactivity in t-glucosyl residues probably represents chain terminations. This labeling pattern is similar to glucan synthesized in the presence of glycerol. The ratio of labeled 4-linked to 3-linked glucosyl residues may increase in the glucan synthesized by the total homogenate as compared to the solubilized enzyme preparation (data not shown). Since different linkages are usually formed by specific enzymes these results indicate that at least two UDP-glucosyl .oowuocooom cow :o>_m o cow auw>wuooowooc wo po>o— —o=uoo on» omoococw ou .o_nwmoow mm: mm nose on .oouocuooocoo mo: .oPQEom oonao>wcoo zoom .Eoo ooo.om op oom.H Eocw oomcoc :owuopxzuoa ocowoa .mopoEom cw auw>waooowooc .ouou on» .ooprooo ocoz mcowuocoooco os>~co ouocooom oz“ cow muooooco :owuoooc ogw .moocuos coo —owcouoe o5» cw ooowcomoo mo ago ma oouocooom mo>wuo>wcoo ozu oco nova—xzuoEcoo oco: muooooco o—oouwowoocoipocmsuo ocw .Axommo coo :wouoco wo a: ow cosy mmo_ .oawu :owuoooc owe cw .omoo:_aimuefigiao= o_oE: No.o\_u: H.o .opoEom coo cowuocoooco oea~co oozpwo wo .3 oomv moowuwocoo xommo ocoocoum coooo oocoooco ocoz Amvcoozpm o>wuooowooc wo mo_oEom Nim o_now 90 o.~ owm omH.H mm m.m we cow OH m.m omH omm om m.H co cm i Amuoooocoooxcwpio o» -mv flxwoo=_m ooxcwpio —amoo:_m ooxcw—im pamooopmiu owuoc ooowmoc :ooo cw sou Amvomouoguczm coospm wo muooooco Nim owns» m cowuocoooco H cowuocoooca .ocoxo_m+ N cowuocoooco fl cowuocoooco pocooxpm i monouozucam oooopm oonpwao—om 91 transferases are present in the solubilized enzyme preparation: a 3-3-glucan synthetase and a 4-B-glucan synthetase. It is not yet possible to conclude whether the latter activity is associated with xyloglucan, glucomannan or cellulose biosynthesis. 0. Preliminary Experiments to Isolate Individual Glucan Synthetases. 1. Affinity Chromatography on Reactive Blue Agarose. The blue chromophore linked to this agarose matrix may be used as a general affinity ligand to purify enzymes which utilize nucleotides or nucleotide derivatives. In two separate experiments, a sample of the solubilized glucan synthetase was applied to a 3 ml column of Reactive Blue Agarose, equilibrated with cholate-glycerol buffer. The sample was held on the column for 45 min. then washed with five volumes of buffer. Subsequently the buffer was supplemented with UDP-glucose (final concentration 1mM) and the enzymes were eluted with 6 ml of buffer. Enzyme activity was monitored for 0.5 ml fractions; glucan synthetase activity eluted in the void fractions, but no enzyme activity was detected in fractions specifically eluted with UDP-glucose. Approximately 85% of the protein applied eluted in the void volume; the remaining 15% specifically eluted with UDP-glucose, but did not exhibit glucan synthetase activity. Thus this affinity procedure does not seem promising as a purification technique: The amount of protein, presumably enzymes other than the glucan synthetases, removed from the enzyme preparation (i.e. bound to the Reactive Blue Agarose) does not give a significant improvement in the specific activity of the sample. 92 2. Gel filtration chromatography. A sample of the cholate- solubilized glucan synthetase preparation was chromatographed on Ultragel ACA34 in buffer containing cholate and glycerol. The fractionation range for this gel matrix is approximately 20,000 to 350,000 daltons for globular proteins. All of the protein eluted in the void volume of the column, indicating that the protein-cholate micelles are in excess of 350,000 daltons (approximate molecular weight). This suggests the membrane bound enzymes are inherently quite large, perhaps part of multi-enzyme complexes. During the gel filtration procedure, enzyme activity decayed significantly. This may reflect losses of cofactors or glucan primers, retarded by the gel filtration column. In addition gel filtration chromatography was performed on Sepharose 28, which has a fractionation range of 200,000 to 2 x 105 daltons. In the initial experiment, no appreciable quantities of protein eluted from the 75 ml column. Sepharose adsorption of lipid and proteins has been observed previously (P. Kelly, personal communication; Klein and Delmer, unpublished results). However, if the same column is used repeatedly, eventually it becomes saturated with lipid and protein and begins to act as a gel filtration matrix. In a second experiment with the same column, the A280 elution profile exhibited two small absorbance peaks well within the included volume of the column, and two peaks of glucan synthetase activity were identified. 93 E. Other Properties of the Solubilized Enzyme Preparation. Additional experiments examined the kinetics of UDP-glucose incorporation by the solubilized enzyme preparation. At a substrate concentration of 0.1mM UDP-glucose (the standard concentration for routine assays) the reaction exhibits roughly linear incorporation kinetics for at least 15 min (with 30 ug of protein per assay; Fig. - 2-5). Between 20 and 60 min., incorporation proceeds but at a decreased rate. The 14C-incorporation was highly variable at later times. Two linear rates may represent the different reaction kinetics of the multiple glucan synthetases in the soluble enzyme preparation; however, an alternative explanation is that the substrate concentration decreased significantly thus slowing the reaction. The initial velocity was measured for substrate concentrations ranging from 1.85 x 10'6 M to 1 x l0"3 M (data not shown). The data was plotted using a variety of standard methods for kinetic analysis, however no meaningful values for the Michaelis constants, Km and Vmax were obtained. The enzymes appeared to exhibit substrate activation, as has been previously demonstrated for glucan synthetases in cotton (Delmer gt al. 1977). In as much as there are at least two glucan synthetases in this preparation, it is perhaps premature to pursue such kinetic analysis. Figure 2-5. 94 Time course of product formation by a solubilized preparation of glucan synthetases. 0.2 ml samples of the solubilized enzyme preparation (containing 29.7 ug of protein) were incubated at 25°; UDP-[14CJ-glucose was supplied to give a final concentration of 0.1 mM, specific activity 5 uCi/umole. The radioactivity in the ethanol precipitable products ranged from 100 cpm (zero time) to 42,000 cpm. Each time point is the average for duplicate samples. 95 m m 522.. oExoowocoocoos 8820 no.2... 60 minutes Figure 2-5 96 Discussion Glucose-containing polysaccharides represent a significant proportion of the extracellular polysaccharides produced by plant cells ,(see Chapter one). Membrane preparations which utilize UDP-glucose to synthesize 3- and/or 4-B-glucans have been described for dicots (e.g. Phaseolus aureus, Gossypium, Lupinus, Pisum) and monocots (e.g. £2222: £91123) (see ref. cited in Delmer, 1977; Darvill 33 al. 1980; Robinson, 1977). However the modulation of these enzymes, whether during cell wall development or regeneration is poorly understood (Delmer, 1977). The UDP-glucose: e-glucan synthetases of soybean cells are primarily associated with a crude membrane preparation (500 to 30,000 x g pellet, Table 2-1). Enzyme activity requires 400 mM sorbitol, possibly as an osmotic stabilizer (Fig. 2-1). This osmotic effect has been observed in a particulate glucan synthetase system derived from Lupinus but is generally overlooked when the glucan synthetase activity is used to identify membrane systems on sucrose gradients (Ephritikhine .££.El-: 1980). Total glucan synthesis in the soybean system is enhanced at alkaline pH; this suggests at least some of the enzymes involved may be localized in the cytoplasm which is normally alkaline. Several detergents with different ionic properties were screened for their effect on enzyme activity and potential to solubilize the soybean glucan synthetases. Only cholate, an ionic detergent stimulates glucan Synthetase activity in the crude membrane preparation and solubilizes appreciable amounts of glucan synthetase (Fig. 2-2). By raising the pH of the buffer system to 7.8, (the pKa of cholate), 97 75% of the enzyme is solubilized after 15 min of detergent treatment (Fig 2-3). The level of glucan synthetase activity in the soluble enzyme preparation, appears to be dependent on the concentration of protein in the assay mixture i.e. enzyme activity is not a linear function of protein concentration. A similar effect was observed for the 3-3-glucan synthetase activity, in a particulate system derived from developing cotton fibers (Heiniger and Delmer, unpublished results). In contrast, the glucan synthetase system from Phaseolus exhibits a linear relation between total glucan synthetase activity and protein concentration (Chambers and Elbein, 1970). When glycerol (final conc. 16.5%) is added to the solubilized enzyme preparation from soybean cells there is a dramatic stimulation of glucan synthesis (e.g. 40 fold, Table 2-1). Glucan synthetase activity recovered in this soluble preparation, measured at low protein concentrations, in the presence of glycerol, represents 80% of the soluble ) total activity (Cf'soluble + pellet The 3-a-glucan synthetase activity of PhaSeolus rapidly decayed at 0°C (Feingold 33 al., 1958). However, I have observed that the solubilized glucan synthetase from soybean cells is stable in the presence of 33% glycerol for at least 24 h. at 4°C and for at least five weeks at temperatures below < 40°C. Some reports have indicated that membrane preparations from Phaseolus (and a digitonin solubilized form), synthesized exclusively 3-3-glucans (Chambers and Elbein, 1970; Feingold 35 31., 1958; Flowers t al., 1968). In contrast, methylation analyses of the glucan products from the soybean system indicate that the soluble enzyme 98 preparation contains at least two glucan synthetase activities since both labeled 3-linked and 4-linked glucosyl residues are detected. In preliminary experiments the ratio of 3- to 4-linked glucosyl residues in the soybean products varied considerably (1.5 to 3) but the distribution of label in the products was not influenced by the addition of glycerol to the reaction mixture. In contrast only the glucan products synthesized by particulate preparation from Lupinus in the presence of glycerol, are susceptible to 3-3-glucanase (from Rhizopus arrhizus (QM 1032); Thomas and Stanley, 1968). Cholate-mediated solubilization and glycerol stabilization of soybean glucan synthetases provides convenient starting material for the isolation of the individual enzymes. This work is now in progress in collaboration with 8. Mitchell in Dr. Delmer's laboratory. In addition we plan to characterize the reaction products: Are the 3- and 4-linked glucosyl residues part of the same polymer or two distinct polymers? Is the 4-3-glucan synthetase activity associated with xyloglucan synthesis or glucommannan synthesis (e.g. is the incorporation of glucose from UDP-glucose stimulated by GDP-mannose or UDP-xylose)? The next chapter will describe some interesting regulatory properties of the 3-3-glucan synthetase, i situ. CHAPTER 3. PROPERTIES OF THE 3-B-GLUCAN SYNTHETASE ifl_situ Results This chapter examines some preperties of the 3-s-glucan synthetase 1 situ. The purpose of these studies is to: I) examine the hypothesis that enzyme activity is controlled, in part, by the localization of the enzyme within the plasma membrane, with the enzyme's active site oriented either towards the periplasmic or cytoplasmic surface (as described in the introduction) and 2) identify other factors which may regulate enzyme activity. A. Glucan Synthesis from Exogenous UDP-Glucose 3-B-glucan is not detected in appreciable quantities in the cell wall or excreted polymers derived from liquid-suspension cultured soybean cells (Figs. 3-1A and 1C). However when whole cells are incubated in the presence of UDP-[14CJ-glucose, the exogenous nucleotide sugar is incorporated into glucan (Table 3-1). This supports the idea that the 3-3-glucan synthetase may accept exogenous substrate but is somehow blocked from utilizing cytoplasmic UDP-glucose. Glucan synthesis is stimulated 3 to 4 fold by plasmolysis. Plasmolysis of intact cells mimics some properties of the physiological state of protoplasts (Boffey and Northcote, 1975). Protoplasts also utilize the supplied nucleotide sugar as a substrate 99 100 for glucan synthesis (Table 3-1). Total incorporation is similar to that of the normal (e.g. not plasmolyzed) cells perhaps because the protoplasts as opposed to the plasmolyzed cells have been purified to remove leaky or severely damaged cells (e.g. caused as a result of the initial plasmolysis). Total incorporation of UDP-glucose varied up to two fold between experiments and represented 0.5 to 1.0% of the supplied 3 UDP-glucose. In several similar feeding experiments when protoplasts were supplied UDP-glucose, less than 0.05% of the label became associated with soluble cytoplasmic pools during the three h. of incubation. This suggests that the plasma membrane is not permeable to UDP-glucose and that the nucleotide sugar is not actively absorbed (also see Delmer £3 ‘31., 1977). After 3 h. less than 1% of the supplied label, remaining in the cultured medium was degraded indicating that enzymes which hydrolyze nucleotide sugars are not secreted into the culture medium. This observation also shows that under these incubation conditions there is a low level of protoplast lysis, which would result in a release of these hydrolases into the culture medium. Total incorporation of exogenous UDP-glucose is increased five fold when prot0plasts are disrupted by osmotic shock, and then incubated in a medium with a water potential equivalent to that of the original protoplast culture medium (Table 3-2). Protoplasts also synthesize significant quantities of 3-8-glucan from supplied 14C- glucose (Figs. 1-78 and 7D). This most probably results from incorporation of [14CJ-gluose into endogenous UDP-glucose pools, after the supplied label had been absorbed (Carpita and Delmer, in press; Karr,_1976). From other experiments (not shown) 101 zum .o cowuoocw poo—oouoco .u mcoeapoo oouocoom .m oppoz ppou .< .omoo:_mimu Huiaoz vow—ooom coon on; cows; mumopoouoco Eocw oo>wcoo mcoexpoo oo—ooo_ cw xuw>wuwoowooc wo o—wwoco :owp:_o ozw .o one u .Amia mww oomv ooaoowocw mw mooowmoc _xmooo_m ooxcw_im wo mo>wao>wcoo on“ wo ocouocooaou cowu:_o ozw .mppoo coooxom ooc:u_:o Eocw oo>wcoo mocoocoum ououooo _ouwopo noun—agnoscoo wo opwwoco cowuo—o mow .m use < .omooapmioo: mooooomoxo Eocw oonmogucxm oooopm wo cowuoowwwucoow .H-m oc=m_c 102 Go 22285.: E 222853 I” .953 .._ om. no. o: .9. o. om. no. o: m... . . a . q a r . 1 m=flv q = 0 .oo. .8. M O -OON -OON m . w ; .oom c -oom oLoov om. 8. o: o. m: m m m w m m H W we .m c < 103 it can be concluded that this synthesis of 3-3-glucan is not the result of gg.ggyg synthesis of the 3-B-glucan synthetase since it occurs without a detectable lag. Taken together the results with protoplasts also support the hypothesis that supplied UDP-glucose is probably utilized at the plasma membrane. The glucan product synthesized under these conditions is primarily 3-B-glucan: 1) Methylation analysis of labeled material derived from protoplasts indicates that the glucose moiety from UDP-[14CJ-glucose is incorporated primarily into 3-linked glucosyl residues (Figs. 3-18 and 10). 2) Samples of the labeled material are extensively degraded by a 3-B-glucanase preparation from Rhizopus suggesting that glucosyl residues are linked in the 8 configuration. A small pr0portion of the label, usually less than 10%, is found in 4-linked glucosyl residues. This 4-linked product is hydrolysed by AN reagent and therefore it is not crystalline cellulose. The 4-linked glycosyl residues may represent low molecular weight precursors to cellulose or other cell wall glucans. Thus, under conditions where protoplasts are competent to synthesize cellulose from 14C-glucose (see chapter 1), UDP-glucose, supplied to the periplasmic surface of the plasma membrane, is not utilized effectively as a substrate for cellulose synthesis. 8. Orientation of the 3-B-Glucan Synthetase in the Plasma Membrane The orientation of the active site of the 3-3-glucan synthetase in the plasma membrane, cytoplasmic versus periplasmic, may control utilization of endogenous versus supplied UDP-glucose. In order to examine the orientation of the enzyme in the plasma membrane more 104 .mucoewcooxo coguo coow umooH Ho cw oocwouno ocoz muHomoc conEHm .oHoE:\_u: m.o wo xuw>wuoo owwwooom a an .25 H wo :oHuococoocoo Hooww o o>wm oa oowHooom mo: omooaHmimoeHHiaos “sowoos wo HE m cw ooocoomomoc ocoz mono—oouoco mcH x ¢.o .czogm pcoawcooxo on» cH .mcowuwocoo coHHEwm cooc: oouoooocw ocoz mama—oouoco .; m cow ocsuocoosou Eooc no oouooaocw ocoz moHosom ogw .oHoe:\_un H wo xuw>wuoo owwwooom a now: 2E H wo cowuocucoocoo Hocww o o>wm ou now—oozm mo: omooz—minuvHuioca .Houwocom Hoosuwz a: once anoos oEom one co .mooguoz woo Howcouoz cw oonwcomoo Eowooe ocouHao umoHoouoco wo .5 m cw ooocoomomoc oco oowzu oogmoz oco: mHHoo osw .HmHHoo NoH x H.H aHouoawxocooov osoHo> HHoo ooxooo .HE m.o oocwoucoo mHHoo oHo Hon N wo moHoEMm Him opnop 105 mumoHoouocm co m—Hoo moH x H s m cw oouocoocoocw moHosc Hugo» moHoEom ouoowHoomo oom.em coo.Nmm ooo.mNN oom.m~ ooo.om omc; m :w oouocoocoocw 5mm .mumoHooaoco oco mHHoo oHosz x2 omooaHmimueHuiooa mzocomoxo wo :owuonHHua .Him oHnoh moon—oouoco mHHou ooNHHoEmoHo mHHou Hocucou 106 .; H cow ocauocooeou Eooc Ho oowccoo ocoz mcowpoazocH .oHoE:\woch wo muw>wuoo owwwooom o saw: .22 H.o wo cowuocucoocoo Hocww o o>wm ou vow—ooom mo; omoozHaumungicoz .moHoEom Hocucoo oz» wo pogo op acoHo>waoo mo: moHoEom on» wo Howucouoo cope: Hooww osu Hosp om cowusHom cowwon oouocucoocoo o sow: ooxwe cos» .xoocm owuoEmo Ho .oomxH umcww ocoz mumoHoouoco wo cooszc acoHo>wzoo c< .Houwczuxcooucoo ze ooH coo NHoou ze m.o .eommz E m .3 E 82-8.: 2... 2 .88 8.. :22... SN 22:28 8:2 we E 8:5 E 885838 ocoz mumoHooHoco ogu .mHocucoo on» :H .mumoHoouoco coH x m.N wo ucoHo>w=oo ogu oocwoacoo moHQEom Nim oanw 107 H.n N.m v.H w.H mama—ouoco on x H coo moHoe: Houow .mumo—oouoco moHoEom ouoowHooco coco.w¢m coco.mo¢ coco.HN moom.ww g H co>o oouocoocoocw sou vow»— Hocucoo wo mwmxH ooHHocazoo an omooonimueHuiooz moocoooxo Eocw mwmocucxm onus—m wo :owuoHaswam Nam open» 108 closely, I attempted to isolate membrane vesicles of uniform topology ((1) membrane vesicles in which the topology remained unaltered; that is membrane vesicles with the periplasmic face remaining outward and (2) inverted vesicles), and examined how these vesicles utilized supplied UDP-glucose. The results are described below. 1. Isolation ofgputative right-side—out plasma membrane vesicles by affinity chromatography. A crude preparation of membrane vesicles was prepared from protoplasts and fractionated on a con A-Sepharose affinity column, as described in the Materials and Methods. Inside-out plasma membrane vesicles, (cytoplasmic face outward), other cellular organelles and excess right-side-out plasma membrane vesicles (periplasmic face outward) should not be retarded on the column. Right-side-out plasma membrane vesicles, with exposed glycosyl receptors, are expected to bind to the column (Brunner gt al., 1977, Glimelius £3 31., 1978; Williamson gt al., 1976; Zachowski and Paraf, 1974). Subsequently these may in principle be specifically eluted from the column with a-methylmannoside. The initial goal was to determine whether a membrane vesicle fraction could be isolated from protoplasts this way, and if so, whether the glucan synthetase in the putative right-side-out plasma membrane vesicles could directly utilize UDP-glucose. The absorbance profile at 280 nm (protein + light scattering by vesicles) of a typical fractionation is shown in Fig. 3-2. The fractions were pooled as indicated and assayed for glucan synthesis from supplied UDP-glucose. The results are summarized in Table 3-3. There is considerable loss (ca. 90%) of glucan synthetase activity during the fractionation procedure; this is presumably due to the 109 Figure 3-2. Separation of membrane vesicles by affinity chromatography on conA Sepharose. Membrane vesicles were prepared from soybean protoplasts and separated on a 2 ml conA Sepharose column as described in the Material and Methods section. The elution profile for material which absorbs or refracts 280 nm light is shown. Fractions were pooled as indicated and assayed for glucan synthetase activity. 110 4.0- 104 t (1 A280 4:- 2.0- |.O- oi-methylmannoside 4.... cholate N I LT Bound I 20 4O 60 “9"“ 3'2 fraction 111 .oouoowocw oco :owuocoooco ocoanoE ooaco mowuHamoc osu one ouomxH umopoouoco Hogwawco ozu wo >9w>wuoo Houop osw .cwe ON cow oomN an “no oowccoo oco: mcowuooom .oHoE:\wu:oH wo Huw>wuoo owwwooom a now: .25 H.o wo cowuocucooooo Hocww o o>wo ou oowHooom mo: omooaHmum zoos—m ocw .Nim .oww cw oouoowocw mm : Ha Nico: Hoozommo mo: cowaoocw sumo ow Auw>wuoo omouozpcxm oo omocogoom <=oo o co oouocooom ocoz moHono> ocoanoz mum oHnow 112 .Hocouocooan Eooc Ho mczo; N xHouoewxocooo couwov mooum HzoocmouoEocgo HHo wo ooo ozu Ho oocHEcoHoo zow>wuoo Hess—oo wooo HooowHo co cow oooHEcouoo auw>wuoot cowuocoooco ococosoa couwo aHouowooEEH ooochouoo xuw>wuoo Howuwcw+ mono o: .o.: .o.: .o.= .o.: mo.o HHH .U... .U.: 4w... mzmv um m.NN H.H OMN Hoo.o H m.NN N.o coo.e owe. oczoo <=oo o.N N.o ONw.H oo.N owo> mcowuoocw esoHoo uoHHoo ~.m N.oH wome.m woo.m m x ooo.oH uoHHoo e.cm mom +ooo.~o mc.c a x ooo.cH acoaoccooom mH.o m.mm +oow.ON o¢.m m x ooo.oH o.m~ NHe +oo~.mm o.eH momma. Heoow chouoco owe CN cw Hommo ocoocoum coo as coo owe coo moHoE:v Ho_>_ooa ocwcooom Hsoocmoooeocgo oouocoocoocw moHoE: Now>cooe Hauoo oouocoocoocw omooon Sou Hmev cwouoco Houow omoconoom wo zuw>wuoo omouosu:»m ooooHo mam o—nmh 113 action of proteases and phenolics compounds in the membrane preparation and the long exposure to room temperature (5 h). In addition fractionations were carried out at room temperature to facilitate conA binding. The bound fraction, putative right-side-out vesicles, utilized UDP-glucose to synthesize ethanol-precipitable glucan indicating that the 3-B-glucan synthetase may accept substrate from the equivalent of the periplasmic face of the plasma membrane. The specific activity of glucan synthetase in the bound fraction is considerably higher than that of the total membrane preparation (measured after affinity chromatography) and-void fraction. This is consistent with the prediction that right-side-out plasma membrane vesicles are preferentially bound to conA. Fraction I exhibited significant glucan synthetase activity. These may represent tailing of the void fraction combined with a slow displacement of right-side-out vesicles from the column. These vesicles are quite large as compared to the size of conA-receptors embedded in the membrane; mechanical forces, including the flow of buffer down the column, may shear the vesicles off of the conA (Brunner _e_t_ 31.,1977). Additional UV absorbing material was eluted from the column when cholate was added to the column buffer (fractions II and III). This may represent very large vesicles trapped at top of the column; cholate would disrupt these vesicles, allowing their constituents to be eluted. Fractions II and III had low and variable low amounts of protein and glucan synthetase activity and were not further characterized. The principal observation - that the putative right-side-out vesicles synthesize glucan from supplied UDP-glucose - is consistent 114 with two models for the orientation of the active site of the 3-e-glucan synthetase in the plasma membrane: 1) The active site is on the periplasmic surface of the plasma membrane. 2) The enzyme, latent lg 1119 (i.e. buried in the plasma membrane) is activated during the preparation of the membrane vesicles and can then utilize supplied UDP-glucose. However these results do not exclude the possibility that the plasma membrane vesicles have become "leaky" to UDP-glucose, with the active site of the 3-8-glucan synthetase on the cytoplasmic face of the plasma membrane. 2. Isolation of putative inside-out plasma membrane vesicles. Experiments were performed to determine whether the 3-8-glucan synthe- tase could utilize UOP-glucose supplied to the equivalent of the cyto- plasmic surface of the plasma membrane. Protoplasts, incubated with latex beads, phagocytize some of the beads; these are now coated with inside-out (cytoplasmic face out) plasma membrane (Hale 33 al., 1980). - €23 .-* co -’ . to €336) - The coated beads are readily isolated by differential centrifugation from lysed protoplasts. In two separate experiments, 3-B-glucan synthetase activity was not detected on the coated beads. This indicates that the 3-3-glucan synthetase cannot utilize UDP-glucose from the cytoplasmic surface; however these experiments are equivocal inasmuch as: i) Low amounts of proteins are isolated on the beads, 115 even under Optimal conditions (Hale gt al., 1980). It was not feasible to measure the amount of protein on the beads because the beads are coated with polyvinylpyrroledine which interferes with the protein assay and ii) Glucan synthesis may require concomitant translocation of the product to the periplasmic space; translocation may be blocked on the beads. C. The Influence of Membrane Potentials on Glucan Synthesis. An electrical potential of roughly 60 to 90 millivolts, negative with respect to the cytoplasm, exists across the plasma membrane of higher plant cells (Rubinstein, 1979). This membrane potential would probably be destroyed when the cell is disrupted. However Carpita and Delmer (in press) suggested that this transmembrane potential may be required to maintain an active cellulose synthetase complex. Recent work by Dr. Bacic in our lab showed that the activity of membrane-bound glucan synthetases from develOping cotton fibers are stimulated four- to 12-fold by treatments believed to induce membrane potentials, which are positive with respect to the inside of the membrane vesicles. Some preliminary experiments were performed to examine this phenomenon in the soybean system to determine whether membrane potential is one factor involved in the regulation of the 3-3-glucan synthetase. 1. Influence of potential on glucan synthesis in membrane vesicles. Protoplasts, disrupted by osmotic shock, were mixed with buffered PEG 4000 to prepare a suspension of vesicular structures (observed by light microscOpy). Membrane potentials were induced in these membrane vesicles by the use of combinations of anions and cations with different permeability coefficients. Thus a positive 116 potential (with respect to the inside of the vesicle) is established by the addition of K+ (SOmM final concentration) in the presence of a K+ specific ionophore, valinomycin, in combination with a relatively impermeant anion MES or $04= (Higinbotham gt al., 1967; Kasai and Komentani, 1979; Pressman, 1976). Valinomycin facilitates transport of K+ down its concentration gradient. A negative potential, with respect to the inside of the vesicle, is induced by combining a relatively impermeant cation (e.g. Bis-tris-propane, BTP+) with a relatively permeant anion, N03‘ (Higinbotham gt al., 1967). Crude membrane vesicles utilize UDP-glucose as a substrate for glucan synthesis (Tables 3-4). A positive potential stimulates glucan synthetase activity by 2 fold (in similar experiments with KMES/val, the activity was stimulated by 50%). The negative potential also stimulates glucan synthesis although the magnitude of the stimulation is smaller. Since the experiments were conducted with crude membrane vesicles it was not possible to measure directly the potentials. If the observed stimulation is due to a transmembrane potential, then replacing the relatively impermeant anion (MES, 504‘) with a more permeant species (SCN’) should diminish or dissipate the potential and decrease glucan synthesis. Less enzyme activity is observed under these conditions (Table 3-4). DCB, a specific inhibitor of cellulose synthesis, slightly stimulates glucan synthesis as compared to the control. However, the stimulation of glucan synthetase activity by the positive potential is not influenced by the addition of DCB (Table 3-4). Glucan synthesis increases slightly (21% c.f. controls) in the presence of ionophores, 117 .ouoowHooo cw ooEcowcoo mo: zoomo :oom .Hocoguo wo mosaHo> N wo cowuwooo on» on oouocHEcou oco owe mH cow ocsuocooeou Eooc Ho ooHoosozw ocoz moowuoooc ooh .AHE H o5=Ho> :owuoooc Honouv oHoE:\wu: m wo Auw>wuoo onwooom o saw: .25 H.o wo cowuocucoocoo Hocww o o>wm ou vow—oozm mo; omoozHoimueHuiooa .oHoEom :ooo ou ooooo ocoz mocogocow oco muHom ouowcoocooo onu oco cwouoco wo a: com xHoHoawxocooo mcwowoucoo muoooHHo HE w.o oucw ooow>wo mo: moHowmo> wo cowmcoomom mcwuHomoc ogw .ouomxH o5» ou ooooo HHHoaooco ocoz coco woo :5 on oocowwoa wo mossHo> xwm .cwE oocgu couw< .ecmmz :2 m .Hco :5 H .¢.H Ia .eom.awm :5 oH .oHoo oocw cocoaHHo Hp oomHH oco: momeHooooca e-m oHneH 118 .o.m mH Hmo.o cmHH Ho>oH em ago He oococowwHo HeeonH=QHm HmeoHn moHosom ouoowpooo wo oooco>\zomc Ho> em.H em.H o.H~ coo.HoH \moo\¢om~¥ HH.H eN.H o.cH oom.¢o Hz=mo moo No.H mo.~ o.o~ coo.moH He>\eom~¥ H25mmo H¢.H o¢.H H.HN ooe.cc «om x oH.H mN.H H.HH occ.eo Hz=mv He> mo.o N.eH coo.Hm Hocoeou :wouoco as coo HimEHizwe moHoE: HHocacoo\uooEHoocHH :HE mH cw moHoE: Hommo coo ouoc wa>waoo wo owuoc xuw>wuoo Houou oouocoocooow sou acoEuoocw elm opnmh .mooocoeoe cooozom Eocw oo>wcoo moHono> cw mHmosucxm cmoonio co mHowucoHoo ococoEoEmoocu oooocw ou oocmwmoo mucosaooco wo Hoowwm 119 .Hzowuoowzoesoo Hozomcoo .coszo .o oco comes: .< .ooou .N.zv moow wo mzowuocuzoommolmeoHHooocazw oouoewumo coo HmNoH .couzoom ozo Hoomv HHHszooscoo o>wuoHoc oouoswumo .HwomH..Ho Ho Eozuozzwmwzv uooc woo ozo moo cow ooszcopoo mouoc ucoomzocu cow co oomoz couowooco mo: acoEHoocH zooo an ooooozw Howuzouoo :H omzozo ozw .oooe mo: zowpo:_5couoo ozo tzo ocoz: .HcoeHoocu Hox oz» cow Hoooxo .zo>wa ow zowuocoocoocw omoco>o ozu coo ouoowHowcu cw ooEcowcoo ocoz mxomm< .Hozozuo wo oHSHo> co>om E 333853 oozoZow .22. o.H cowuocuzoocoo Hozwwv ooooo mo: omooszuooa ooHooncz .zwe ON cow ocsuocoosou sooc no oooozoozH oco: moHoEom ozw .oHoE:\wuo o wo xuw>waoo owwwooom o zuwz :5 H.o wo zowuocuzoocoo Hozww o o>wm o» oHoEom zomo ow ooooo mo: omoooniooa .mmz.owm cow zowuwooo oz» Hz .coHoEmowHHwe com wo uono>wooo ozH Ho acoumzoo oHoz mo: acoEuoocu zooo wo Howuzouoo cope: oz» Hooumowocw mo .m.m :o .cowwoz wo HE m.o ow ooozoomomoc ocoz memo—oouoco ozw .szouoco mo meV mama—oouoco moH x m oocHouzoo moHoEom mum onow 120 H25 oomw Ne.H Hc.H oeH.N oNHceHoooo .=_ Hm>\¢om mo.~ N¢.m oo¢.m oNHcaHoooo .eH Hm>\zomc :5 omN Ne.m H.o on.o Hoocwo o: co oNHceHoooo .=_ zome :5 omm om.H om.H ooe.~ oNHcmHoooo .=_ He>\Hoc :5 0mm Ho.m No.8 owo.o oNHceHoaoe co Hoocco 0: .=_ Hoe mm.c oo.o ooc.H oNHceHoocoocz .Hao H~>\moz-ecm HHocHzoov - o~.H o~¢.H Hoowco o: .ocoe moz-ecm onw +¥ AHocuzoo\uzoEuoocuv Owe ON cw zuw>wuoo zwouoco Os coo moHoE: muooooco coon—O Howuzouoo ozocoEos oEHNOo owuoc cowuocoocoozw Hopoa zw Eoo Houoa OH omzmzo oouowooco pooEHoocw o >w>_mw mwmozuoam zoos—O :o Howuzouoo ozoczsoe couHo op oocawmoo mucosaoocu wo oozoonzH mim o—OOP 121 without K+ (Table 3-4). Valinomycin, and DCB are hydrophobic compounds which become associated with the membrane and thus may alter membrane environment resulting in a slight stimulation of glucan synthetase activity. The glucan products, examined by methylation analysis, contain both 3-linked and 4-linked glycosyl residues in an approximate ratio of 10 to 1. A preliminary experiment indicates this distribution of products is not altered by the induction of a positive potential in the vesicle preparation. The glucan products are solubilized by AN reagent, thus the labeled 4-linked glucosyl residues do not represent crystalline cellulose. 2. Influence of membrane patential on 3-8-glucan synthesis, from exogenous UDP-glucoseg in vivo. Experiments were designed to examine whether pertubation of the transmembrane potential of protoplasts would affect glucan synthesis from exogeneous UDP-glucose. Treatments were designed to establish K+ flow in or out of the protoplasts by incubating protoplasts with valinomycin and/or in the presence of high levels of K+ salts (Table 3-5). K+ was combined with anions having different permeability coefficients; it is not clear as to whether these treatments may have other indirect effects on enzyme activity. When the membrane potential is hyperpolarized, glucan synthesis is inhibited. This effect is also observed when protoplasts are incubated in high concentrations of BTP-N03 (e.g. hyperpolarized, data not shown). K+ combined with the relatively impermeant anion 504‘, is expected to depolarize the protoplasts, this effect should be facilitated by valinomycin. Glucan synthesis is stimulated by this treatment (Table 3-5), however the degree of stimulation is higher when 122 K+ is combined with a relatively more permeant anion (Cl‘ or SCN'). Furthermore the addition of valinomycin actually decreases glucan synthesis (cf. KSCN treatment 3 val). The results from these in vivo experiments do not conform to a simple model by which transmembrane potential regulates 3-8-glucan synthetase activity: Total glucan synthesis does not reflect the predicted change in membrane potential. As indicated earlier the effects of these treatments on other aspects of cellular physiology may have indirect effects on 3-B-glucan synthetase activity. Nonetheless the imposition of a transmembrane potential on membrane vesicles from soybeans stimulates glucan synthesis. This implies that the transmembrane potential may have a regulatory function in the synthesis of 3-3-glucan. 123 Discussion 3-B-glucan is not found in appreciable quantities in the walls or excreted polymers from liquid suspension cultures of soybeans. However it is synthesized in significant amounts from exogeneous UDP-glucose during the early stages of wall regeneration and also by plasmolyzed cells. These observations may indicate that the plasma membrane has become "leaky“ to UDP-glucose, thus the supplied nucleotide sugar diffuses to the active site of the 3-3-glucan synthetase on the cytoplasmic surface of the plasma membrane (see the models described in the Introduction). However, that model does not explain why under normal conditions the enzyme does not utilize endogenous UDP-glucose. Nhat discriminates between these substrate pools jg 1122? Alternatively if the active site of the enzyme is localized on the periplasmic surface of the plasma membrane, how is enzyme activity stimulated by protoplast lysis? Is some latent form of the enzyme activated by wounding (Brett, 1978)? Is the mechanism(s) of enzyme activation a function of membrane perturbation? In order to elucidate the mechanism(s) of 3-s-glucan synthetase regulation 1_ vivo experiments were untaken to examine the orientation of the enzyme in the plasma membrane. Orientation of the 3-3-Glucan Synthetase. Hughes and Gunning (1980) have proposed that the active site of the 3-3-glucan synthetase is on the periplasmic surface of the plasma membrane. They have suggested that when a cell is wounded, i.e. the integrity of the plasma membrane is disrupted, cytoplasmic UDP-glucose diffuses to the active site of the enzyme and the resulting synthesis of 3-B-glucan forms a "plug" at 124 the damaged site. This model was developed from cytological observations of the formation of callose plugs around plasmodesmata. Anderson and Ray (1978) reported that when UDP-glucose was supplied to pee stem sections, it was utilized primarily at cut edges for the synthesis of 3-e-glucan. Mueller and Machachlan (1980) prepared microautoradiograms of pea stem sections which had been incubated with labeled UDP-glucose; in these samples the plasma membrane was appressed to the cell wall and the periplasmic surface of the membane was not examined. Incorporation of UDP-glucose appeared to occur at the cytoplasmic surface. The resolution of this type of microautoradiograph is comparatively low and thus these results do not exclude alternative models for the orientation of the enzyme in the plasma membrane (e.g. both cytoplasmic and periplasmic orientation, Montezinos, personal communication). Affinity chromatography on conA Sepharose was used to isolate putative right-side-out plasma membrane vesicles derived from soybean protoplasts. Glucan synthetase II (3-B-glucan synthetase) and a K+-stimulated ATPase, are the only markers so far suggested for the plasma membrane of higher plants (Leonard and Hodges, 1980; Quail, 1979). The ATPase is fairly labile (C. Caldwell, personal communication). Mitochondria of mammalian cells are known to contain conA receptors (Bittiger, 1976); however, it seems unlikely that their counterparts in plants contain 3-8-glucan synthetase activity (Leonard and Hodges, 1980; Quail, 1979). In the absence of more refined techniques to characterize the vesicles fractionated on conA Sepharose as plasma membrane vesicles, tentative interpretation of these experiments provides some new information with which to evaluate 125 various models for the orientation of the 3-3-glucan synthetase in the plasma membrane: UDP-glucose supplied to the equivalent of the periplasmic surface of the plasma membrane serves as a substrate for glucan synthesis. The Influence of Membrane Potential on 3-B-Glucan Synthetase Activity. When the integrity of the plasma membrane is disrupted, (i.e. during wounding) 3-e-glucan synthesis is stimulated. This may, in part, be the result of enzyme activation (Brett, 1978), e.g., the physical environment of membrane bound enzymes may influence enzyme activity (Dupont, 1979; Helenius and Simons, 1975). Treatments designed to induce of a positive potential across soybean membrane vesicles derived from protoplasts, results in a 1.5 to 2 fold stimulation of glucan synthesis from supplied UDP-glucose. This potential may influence enzyme conformation within the membrane or alter the membrane environment (e.g. fluidity) thereby indirectly influencing enzyme activity (Dupont, 1979; Laane gt al; 1979, Lelkes; 1979; Toci gt al., 1980). Alternatively, the electrical gradient may facilitate the movement of the negatively charged substrate across the‘ membrane to the active site of the enzyme. This last interpretation is perhaps less likely: Imposition of a negative potential also results in a stimulation of glucan synthesis, albeit smaller than that of the positive potential. These observations actually suggest a paradox: Membrane depolarization (i.e. protoplast lysis) results in a stimulation of 3-B-glucan synthetase activity. Treatments designed to impose a positive or negative potentials across the isolated membrane vesicles 126 also stimulates enzyme activity (i.e. membrane polarization). Both effects implicate membrane environment as a regulatory factor in 3-B-glucan synthesis. These are perhaps related to the mechanism by which mild temperature stress induces callose synthesis in corn (Smith and McCully, 1977). In addition elucidation of the mechanisms of 3-8-glucan synthetase regulation may help identify factors which regulate the other plasma membrane glucan synthetase, the cellulose synthetase (Delmer, 1977). CONCLUDING REMARKS Cell wall regeneration by soybean protoplasts provides a good model system for studying the many facets of cell wall biogenesis. In contrast to protoplasts isolated from leaf tissue of dicots and tissue cultures of monocots, soybean protOplasts rapidly begin to synthesize the fibrillar components of the primary cell wall, i.e. cellulose, when transferred to suitable culture medium. In addition, within three hours of wall regeneration, protoplasts synthesize and secrete other polysaccharides which qualitatively resemble components of the native cell wall and also polymers secreted into the culture medium by suspension cultures (see Chapter one). Because of the relative ease with which protoplasts can be disrupted (as compared to intact cells) it will be possible to isolate cell wall precursors from protoplasts, obtaining new information about their composition and complexity as well as insights into the assembly of these polymers into a cell wall. At least two polysaccharide synthesizing enzymes are part of the plasma membrane and the regulation of their activity may be in part mediated through their localization in the membrane and by the fluidity of the plasma membrane (Chapter three). The exposed plasma membrane of protoplasts may be probed with saphisticated procedures developed for mammalian cells in order to examine how the plasma membrane modulates the activity of these enzymes. Further purification and 127 128 characterization of these enzymes will provide important information about the biosynthesis of cell wall polysaccharides. The influence of membrane potential on enzyme activity can be examined in more detail using solubilized forms of the s-glucan synthetases. In reconstitution experiments the isolated enzymes may be inserted in to artificial lipid vesicles; these can be used to verify that the induced potentials actually influence enzyme activity. FOOTNOTE 1Previously this enzyme and one that utilizes GDP-glucose have both been designated cellulose synthases (E.C. 2.4.1.12 and E.C. 2.4.1.29). These assignments are incorrect, (see below, Darvill ‘gt‘gl., 1980; Delmer, 1977). Many of the other polysaccharide-synthesizing enzyme have not been assigned E.C. numbers). 129 REFERENCES Albersheim, P. (1976) The primary cell wall. In "Plant Biochemistry" Bonner, J. and Varner, J.E., Academic Press, Inc., New York, NY, pp. 225-274. Albersheim, P., Nevins, 0.5., English, P.D. and Karr, A. (1967) A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydrate Res. 5:340-345. Anderson, R. and Ray, P. (1978) Labeling of the plasma membrane of pea cells by a surface localized glucan synthetase. Plant Physiol. 61:723-730. A Asamizu, T. and Nishi, A. (1979) Biosynthesis of cell wall polysaccharides in cultured carrot cells. Planta 146:49-54. Asamizu, T., and Nishi, A. 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