Biosynthesis of Cellulose*

ADVANCES IN CARBOHYDRATE CHEMISTRY AND BIOCHEMISTRY, VOL. 41

BIOSYNTHESIS OF CELLULOSE*

BY DEBORAH P. DELMER MSU-DOE Plant Research Iaboratory, Michigan State Uniuersity, East Lansing, Michigan 48824**

................................................. 105 . . . . 107 . . . . . . . . . . . . 110 IV. Cytological Investigations of Cellulose Biosynthesis . . . . . . . . . . . . . . . . . . 116 1. The Site of Cellulose Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2. Orientation of Microfibrils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .123 V. The Mechanism of Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 1. Involvement of Glycosyl Esters of Nucleoside Diphosphates . . . . . . . . , . 125 2. Possible Involvement of Lipid Intermediates . . . . . . . . . . . . . . . . . . . . . . 132 I. Introduction

11. A Survey of Organisms Useful for the Study of Cellulose Biosynthesis 111. Structural Considerations Relevant to Biosynthesis ... ....

.

3. Possible Involvement of High-molecular-weightPrecursors to Cellulose 135 4. Genetic Mutations, and Chemical Inhibitors of Cellulose Biosynthesis . . 143 5. Possible Factors Affecting the Lability of the Polymerizing System . . . . . 145 VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 VII. Addendum .................................................. 152

. .. . .

..

I. INTRODUCTION

The topic of the biosynthesis of cellulose was discussed by Shafizadeh and McGinnis' in this Series about a decade ago, and since then, several reviews have appeared in the literature?-' One theme com* This article was written while the author was on a sabbatical leave at the Department of Biological Chemistry, The Hebrew University, Jerusalem, Israel. She is grateful for support for this effort from Michigan State University, from the U. S.-Israeli Binational Agriculture Research and Development Fund (BARD),and from The Hebrew University. ** Present address: ARC0 Plant Cell Research Institute, 6560 Trinity Ct., Dublin, CA 94568. (1) F. Shafizadeh and G. D. McGinnis, Ado. Carbohydr. Chem. Biochem., 26 (1971) 297-349. (2) J. R. Colvin, CRC Crit. Reu. MacromoZ. Sci., 1 (1972)47-84. (3) D. P. Delmer, Recent Adu. Phytochem., 11 (1977)45-77. (4) D. G . Robinson,Adu. Bot. Res., 5 (1977)89-151. (5) G. A. Maclachlan, Trends Biochem. Sci., 2 (1977) 226-228. (6) A. Darvill, M. McNeil, P. Albersheim, and D. P. Delmer, in N. E. Tolbert (Ed.), The Biochemistry ofPZants, Vol. I, Academic Press, New York, 1980, pp. 91-162. (7) J. R. Colvin, in J. Priess (Ed.), The Biochemistry ofPZants, Vol. 111, Academic Press, New Yorlc, 1980, pp. 543-570. 105

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0072414

106

DEBORAH P. DELMER

mon to all of these is the lament that, despite the fact that cellulose is the world’s most abundant organic polymer, so little is known about its mode of synthesis. Those working in this field are well aware that the older literature is confusing and contains errors, misinterpretations, and incomplete data. The key to sorting out all of this confusion must come in the recognition that it has been exceedingly difficult to demonstrate convincingly the synthesis of true, microfibrillar cellulose by using cell-free preparations derived from cells that are capable of abundant synthesis ofcellulose in uiuo. As one engaged in studying this process in higher plants, the present author doubts whether this has ever been achieved with cell-free preparations derived from these organisms; and, even in the case of the bacterium Acetobacter xylintina, where convincing synthesis of (l+4)-p-@glucan has been achieved in uitro, the in uitro rates of synthesis are, in general, less than 1% of those observed in z;iuo; and, as will be discussed in Section V , l , it is not even certain that, in all cases, (1+4)-p-@glucan should be equated with microfibrillar cellulose. Such a situation, although frustrating, is also fascinating. Are we supplying the correct substrates in uitro? If so, what is the nature of the extraordinary lability of the cellulose-synthesizing system? What essential features of the intact system are lost when cells are broken, or even just rendered permeable to substrates? The key to unraveling the complexities of the biosynthesis of cellulose will only come when full answers to such questions have been obtained. The importance of the older, more confusing literature in the field is that it established two incontrovertible facts-that the process is undoubtedly complex and that we must look beyond simple conclusions in order to progress further. The real cause for optimism is that these difficulties are at last being recognized, and that the past few years have witnessed a remarkable maturing of the field and have presented us with some intriguing new findings. Elegant visual observations of the process of cellulose biosynthesis by microscopists have confirmed the complexity that the biochemists predicted; new evidence concerning the nature of possible precursors to cellulose has been presented; and, finally, new insights into the reasons for the lability of the system are being explored. The purpose of this article is, therefore, to attempt, first of all, to analyze the older, fragmented literature and assess its major contributions; secondly, to concentrate extensively on new findings, and attempt to coordinate their interpretations; and thirdly, to indicate what gaps in our knowledge of this complex process still exist.

BIOSYNTHESIS OF CELLULOSE

107

11. A SURVEY OF ORGANISMS USEFUL FOR THE STUDY OF CELLULOSEBIOSYNTHESIS

Before discussing the literature on the biosynthesis of cellulose, it may prove useful to describe some characteristics of the various organisms available for study, and to attempt to assess the various advantages and disadvantages presented by each. Among the bacteria, only the genus Acetobacter produces abundant quantities of cellulose. Largely due to the pioneering efforts of Hestrin and coworkers in Israel: Acetobacter xylinum has emerged as a classic organism for the study of cellulose biosynthesis. Unlike the algae and higher plants, the cellulose of A . xylinum is not produced as a cell-wall component, but as an extracellular pellicle of essentially pure cellulose. A . xyh u m is a non-motile, strict aerobe; a teleological argument has been made that the pellicle is produced as a means of allowing the cells to maintain an adequate supply of oxygen by virtue of their continued association with the floating pellicle. This argument is supported by the experimental observation that mutants which lack the ability to synthesize cellulose can be readily isolated from cultures grown under vigorous a e r a t i ~ nThe . ~ advantages of studying A . xylinum are numerous: it is a unicellular organism, and is easily propagated; numerous studies have elucidated the details of its pathways of carbohydrate metabolism (see references cited in Ref. 10); and mutants lacking the capacity for cellulose synthesis can be isolated. As disadvantages, it lacks a well-defined, genetic system, and is far removed on the evolutionary scale from higher plants, the major producers of cellulose. However, insufficient information is as yet available to allow speculation as to the direction and extent to which the synthesizing system has evolved. There are a few reports that indicate that a few other genera of bacteria are capable of limited synthesis of cellulose11J2;some fungi are also capable of synthesis of cellulose,13as well as a few rare members (8) S. Hestrin, in I. C. Gunsalus and R. Y. Stanier (Eds.), The Bacteria, Vol. 111, Academic Press, New York, 1962, pp. 373-388. (9) M. Swissa, Y. Aloni, H. Weinhouse, and M. BenzimanJ. Bacteriol., 143 (1980) 1142 -1150. (10) Y. Aloni and M. Benziman, in R. M. Brown, Jr. (Ed.),Cellulose and Other Natural Polymer Systems: Biogenesis, Structure, and Degradation, Plenum, New York, 1982, pp. 341-361. (11) E. Canale-Parola and R. S. Wolfe, Biochirn. Biophys. Acta, 82 (1964)403-405. (12) M. H. Deinema and L. P. T. M. Zevenhuizen,Arch. Mikrobiol., 78 (1971)42-57. (13) J. M. Aronson, in G. C. Ainsworth and A. S. Sussman (Eds.),The Fungi, Vol. I, Academic Press, New York, 1972, pp. 49-76.

108

DEBORAH P. DELMER

of the animal kingdom, most notably the tunicates.I4 However, in none of these organisms has the synthetic process been studied in detail. Many of the algae synthesize cell walls that contain cellulose. A book by Preston15gives an excellent description of the cell-wall structure of these algae, the most widely studied of which are the genera Valonia, Oocystis, Microsterias, Cladophora, Chaetomorpha, Glaucocystis, and Pleurochrysis. To date, surprisingly few biochemical studies have been conducted with these organisms, perhaps partly because of difficulties in culturing these algae in large quantities, and in breaking the cells. However, some of the unicellular algae have served as excellent models for microscope examinations of the process, which have been facilitated by the unusually large, and highly ordered, cellulosic microfibrils present in their cell walls. Among the nonphotosynthetic eukaryotes, several organisms have emerged as possessing good potential for biosynthetic studies. These organisms are: Prototheca zopfii, a colorless alga having a cellulosic cell-wall; the slime molds Polysphondylium pallidum and Dictyostelium discoidum; and the amoeba Acanthamoeba castellanii. The slime molds and Acanthamoeba produce a cellulosic wall during one specific stage of development (encystment); they may therefore be of use for studies of regulation, and may also have the advantage that relatively undamaged, plasma membranes could be isolated from cells before extensive encystment occurs.16All of these organisms hold special promise, because some in uitro synthesis of (1+4)-P-~-glucan from UDP-glucose has been observed on using cell-free preparations, even though no unique conditions for extraction or assay were emp l ~ y e d . ' ~ The - ' ~ zygote of the brown alga Fucus also produces a cellulosic wall upon fertilization,*Obut in uitro studies with this cell type have not been reported to date. Among the higher plants, much enzyme work has been conducted with elongating, etiolated hypocotyls or epicotyls of such legumes as mung beans (Phaseolus aureus) or peas (Pisum satiuum). These tissues have the advantages of being quite easy to grow, possessing cells (14) A. B. Wardrop, Protoplasma, 70 (1970) 73-86. (15) R. D. Preston, The Physical Biology ofPlant Cell Walls, Chapman and Hall, London, 1974. (16) M. L. Phillippi and R. W. Parish, Planta, 152 (1981) 59-69. (17) H. E. Hopp, P. A. Romero, G. R. Daleo, and R. Pont-Lezica, Eur. 1.Biochem., 84 (1978) 561-571. (18) C. Ward and B. E. Wright, Biochemistry, 4 (1965) 2021-2026. (19) J. L. Potter and R. A. Weisman, Biochim. Biophys. Acta, 237 (1971) 65-74. (20) R. S. Quatrano and P. T. Stevens, Plant Physiol., 58 (1976) 224-231.

BIOSYNTHESIS OF CELLULOSE

109

engaged in active, cell-wall synthesis, and yielding extracts relatively free from pigments and deleterious, phenolic compounds. However, such tissues are composed of more than one cell type, and suffer the disadvantage that a number of other cell-wall polymers are also synthesized at this stage of development. Woody tissues, which are rich in secondary-wall cellulose, but also contain large proportions of lignin, have not been exploited for studies on cellulose synthesis. However, another cell type rich in secondarywall cellulose, namely, the developing cotton-fiber, has emerged as a useful experimental system; the fibers constitute a homogeneous celltype which elongates synchronously with time and, at a precisely regulated time in its development, initiates the rapid synthesis of a cellulosic secondary-wall that is free from lignin. Further advantages are that ( a ) the pattern and composition of the developing cell wall has been studied in detail by Meinert and DelmeP and Huwyler and coworkers,2z (b) the flow of carbon in vivo from D-glucose to end products has been extensively analyzed by Carpita and Delmer,23and ( c ) the fibers can be cultured in uitro, with their associated ovules, by techniques devised by Beasley and Ting24 and Waterkeyn and cow o r k e r ~ ?A~ major disadvantage of this system is that extensive growth-facilities must be maintained in order to ensure a constant supply of flowering cotton-plants. Plant cells proliferating in tissue culture have also been used; here, the cell type is somewhat more homogeneous, but a variety of polysaccharides is produced. A great deal of optimism was held for the use of protoplasts derived from such cells; such protoplasts can often regenerate a cell wall, and it was considered that precursors might be directly fed to such cells, or that membranes isolated by gentle, osmotic lysis might be more active for studies of synthesis in vitro. However, experiments by Klein and DelmeP with soybean protoplasts, and by Haass and coworkersz7with tobacco protoplasts, indicated that such protoplasts, although capable of producing cellulose during wall regeneration in uiuo, are unable to use glycosyl esters of nucleotides directly for cellulose synthesis; and, in general, they have yielded re(21) M. Meinert and D. P. Delmer, Plant Physiol., 59 (1977) 1088-1097. (22) H. R. Huwyler, G . Franz, and H. Meier, Planta, 146 (1979) 635-642. (23) N. C. Carpita and D. P. Delmer,J. Biol. Chem., 256 (1981)308-315. (24) C. A. Beasley and I. P. Ting,Am.J. Bot., 61 (1974) 188-194. (25) L. Waterkeyn, E. de Langhe, and A. A. H. Eid, Cellule, 71 (1975)41-51. (26) A. Klein and D. P. Delmer, PZanta, 152 (1981) 105-114. (27) D. Haass, W. Blaschek, H. Koehler, and G . Franz, in D. Robinson and H. Quader (Eds.), Cell Walls, Wissenschaftliche Verlags, Gmb H, Stuttgart, 1931, pp. 109118.

DEBORAH P. DELMER

110

sults in studies in rjitro with isolated membranes similar to those obtained with other plant cells possessing cell walls. 111. STRUCTURAL CONSIDERATIONS RELEVANT TO BIOSYNTHESIS

No critical discussion of biosynthesis can be attempted without consideration of'the information available on the structure of native cellulose. As for studies on biosynthesis, much controversy has existed in the field of structural analyses of cellulose. There seem to be only two points on which all workers are in agreement, namely, that ( a ) native cellulose is a composite of linearly extended chains of (1-+4)-/3-D-glucan (l),and ( b ) strong intrachain and interchain hydrogen-bonding between D-glucosyl residues occurs in such a way as to create a highly insoluble, and partially crystalline, fibrillar structure.

HO~~o~

o OH

CH,OH

~OH OH

n

CH,OH

Cellulose (1-4)-p-D-Glucan 1

For studies of biosynthesis, even this limited information imposes certain necessary considerations. Any product synthesized in vitro should be chemically characterizable as (l+"i)-P-D-glucan. The presence of small proportions of sugars other than D-glucose has often been detected in samples of "purified' cellulose28*'Y; it is not known whether these constituents are truly a part of the covalent'structure. Even so, the proportions are quite low, and, as such, should constitute only a small fraction of the product. The product synthesized in vitro should be insoluble in alkali, but this criterion alone is not acceptable as proof of cellulose synthesis, as other D-glucans, most notably (1-+3)fi-D-glucans, may also be insoluble under these ~ o n d i t i o n s . 4 *The ~*~~~~* product should be degraded by enzymes specific for the p-~-(1+4)glucosidic linkage, but proof must be given that the enzymes used are pure, and this has rarely been offered. Furthermore, proof should be A. Adams and C . T. Bishop, Tappi, 38 (1955) 672-675. (29) 13. T. Dennis and R. D. Preston, Nature, 191 (1961) 667-668. (30) Y . Raymond, G. B. Fincher, and G. A. Maclachlan, Plant Physiol., 61 (1978) 938(28)

942.

(31) U. Heiniger and D. P. Delmer, Plant Physiol., 5941977) 719-723.

BIOSYNTHESIS OF CELLULOSE

111

given that the series of oligosaccharides released, either by enzymic digestion or by partial hydrolysis with acid are, in fact, cello-oligosaccharides; unfortunately, identifications have usuaIly been based solely on comigration during paper chromatography with cello-oligosaccharide standards, with no indication given as to whether the separation procedure used would allow resolution of a cello-oligosaccharide series from a series of oligosaccharides having a different linkage. Having free, vicinal hydroxyl groups, the product should be attacked by periodate, but the high degree of insolubility creates difficulties in achieving complete reaction. Similarly, methylation analyses should yield 2,3,6-tri-O-methyl derivatives Of D-glucose, but, once again, insolubility often leads to low yields. For cellulose, new solvents that are nondegrading have been employed and have been found to improve the yields The product should theoretically give an X-ray diffraction pattern typical of cellulose I (or cellulose 11, if treated with alkali; see later discussion), but the limited yields to date in synthesis in vitro, as well as the impurity of the preparations, usually preclude this analysis. Thus, even a chemical characterization of the products synthesized in vitro is fraught with difficulties, and this problem is responsible for some of the confusion in the literature on biosynthesis. In Section V,1, the question of whether true, microfibrillar cellulose can be expected as a product of in vitro synthesis will be discussed in more detail. Another problem for biosynthesis concerns the fact that certain other, native plant-polysaccharides, most notably the xyloglucans found in higher-plant cell-walls,6 contain backbone structures of (l-A)-P-D-glucan, and it is necessary to consider whether separate enzymes synthesize these structures, and, if so, whether these enzymes could be confused with enzymes specifically involved in the synthesis of cellulose. Much controversy has existed over the structure of the so-called microfibril. From electron-microscope studies, it is clear that all native celluloses exist in linear aggregates, or fibrils, of discrete size. However, although being well defined and precisely regulated within one cell type, the size varies considerably among different organisms, or even at different stages of development within one cell type. The diameters of the fibrils are various: the smallest fibrils noted have a diameter of 1.5 nm, the so-called “sub-elementary fibril,” which has been seen in wood cambium33and quince slime34;the frequently ob-

-

(32) J.-P. Joseleau, G. Chambat, and B. Chumpitazi, Carbohydr. Res., 90 (1981) 339344. (33) R. B. Hanna and W. A. Cote, Jr., Cytobiologie, 10 (1974) 102-116. (34) W. W. Franke and B. Ermen, Z. Naturforsch., Teil B , Z4 (1969)918-922.

112

DEBORAH P. DELMER

served, 3.5-nm “elementary fibril” that is found in Acetobucter xyl i n ~ r nin, ~the ~ primary cell-walls of higher plant^,'^ and in the frayed ends of larger micro fibril^^^; and diameters of 5-10 nm for the secondary walls of wood:’ 10-20 nm for such fiber cells as ramie:* and, finally, up to 50-200 nm, characteristic of the cellulose in cell walls of unicellular algae.l5 The controversy partly concerns the questions of whether ( a )a fundamental and constant unit, such as the 3.5-nm elementary fibril proposed by Muled1aler,3~exists in nearly all native celluIoses, and ( b ) larger microfibrils are constituted of aggregates of these elementary fibrils. The various models of microfibrillar structure proposed have been well reviewed by Colvin2and by Shafizadeh and McGinnis.‘ Part of the controversy arises from the fact that crystallite sizes observed by X-ray diffraction analyses do not always correspond to fibril sizes observed by microscopy. The presence of amorphous regions in crystalline cellulose also creates problems in interpretation, as was clearly discussed by Shafizadeh and McGinnis.’ At present, it would seem that many workers accept the concept that the 3.5-nm elementary fibril is a very common (although, perhaps, not universal), discrete entity. Packing analyses indicate that such a fibril would contain -36 Dglucan chains. The question of the existence ofa discrete and uniform elementary fibril is relevant to considerations of biosynthesis. Does such a discrete fibril arise as the result of synthesis by a multi-subunit, enzyme complex, with each subunit responsible €or the polymerization of a single Bglucan chain? Although P r e ~ t o n ’ ~ argued against elementary fibrils, he nevertheless recognized that cellulose is composed of many chains, and he was responsible for first proposing4O the so-called “ordered-granule hypothesis” which envisages such a multi-subunit complex embedded in the plasma membrane (see Fig. 17 in Ref. 1).If the basic fibril is the 3.5-nm elementary fibril, such a complex should contain 36 subunits. A proposal by Haigler and Benziman4I for A. xylinum envisages closely spaced, synthetic sites, each containing 12-15 subunits that synthesize 1.5-nm subelementary fibrils, which then self-associate to form the 3.5-nm elementary fibril (see Fig. I). However, based on existing data, it is (35)R. M. Brown, Jr., J. H. M. Willison, and C. L. Richardson, Proc. Natl. Acod. Sci. CJSA,73 (1976)4565-4569. (36)J. Blackwell and F. J. Kolpak, Macromolecules, 8 (1975)322-326. (37)A. J. Hodge and A. B. Wardrop, Nature, 165 (1950)272-279. (38)A. Vogel, Makromol. Chem., 11 (1953)111-117. (39)K. Mulethaler, Beih. Z. Schweiz. Forstu., 30 (1960) 55-62. (40)R.D.Preston, in M. H. Zimmerman (Ed.),The Formation of Wood in Forest Trees, Academic Press, New York, 1964, pp. 169-188. (41) C. H. Haigler and M. Benziman, in Hef. 10, pp. 273-297.

BIOSYNTHESIS OF CELLULOSE

(1+ 4)-p-D-Glucan

113

polymerizing enzymes

FIG.1.-Proposed Model of Cellulose Assembly in Acetobacter ~ylinum.~' [DGlucan chain aggregates from organized,multiple-enzyme complexes, and extrusion pores crystallize into microfibrils, which then assemble into bundles and the normal ribbon at the cell surface.]

certainly not necessary to invoke one discrete, elementary-fibril size for all organisms; fieeze-fracture studies on algae suggested that enzyme complexes of various sizes can exist (see Section IVJ). X-Ray crystallography has contributed greatly to our understanding of the structure of cellulose. It has been known for many years that cellulose can exist in at least four different, crystalline forms termed42 celluloses I, 11, 111, and IV. Essentially all native forms of cellulose a form thermodynamically less stable exist in the cellulose I than cellulose 11; when native cellulose I is dissolved and recrystallized, or subjected to swelling in alkali ("mercerization"), it is converted into the more stable, cellulose I1 structure. For purposes of biosynthesis, the most critical question addressed by these studies has concerned the orientation of the chains in cellulose I, that is, whether all of the chains are aligned parallel, or antiparallel. There seems to be uniform agreement that the chains of cellulose I1 are arranged in an antiparallel o r i e n t a t i ~ n The . ~ ~ original structure proposed for cellulose I by Meyer and M i s ~ also h ~ possessed ~ antiparallel chains. However, more-refined methods of analysis led to re-evaluation of the structure of cellulose I, and indicated that the most favored models show the chains to be parallel. The most definitive work was performed, with the native cellulose of the alga Valonia, by Gardner and Bla~kwell"~ and Sarko and M ~ g g l i , 4and, ~ for ramie fibers, by Woodcock and S a r k ~all ; ~concluded ~ that the chains of native cellulose I are parallel. French4' also extensively examined the X-ray data for ramie (42) A. Sarko, Tappi, 61 (1978) 59-61. (43) K. H. Meyer and L. Misch, Helo. Chim. Acta, 20 (1937) 232-244. (44) K. H. Gardner and J. Blackwell, Biopolymers, 13 (1974) 1975-2001. (45) A. Sarko and R. Muggli, Macromolecules, 7 (1974) 486-441. (46) C. Woodcock and A. Sarko, Macromolecules, 13 (1980) 1183-1187. (47) A. D. French, Carbohydr. Res., 61 (1978) 67-80.

DEBORAH P. DELMER

114

fibers and, although he stated that a parallel model is possible, he concluded that an antiparallel orientation cannot yet be completely excluded. However, the fact that a derivative of native ramie cellulose, namely, cellulose triacetate, has been shown by Stipanovic and Sarkd8 to have the parallel orientation, further strengthens the case for parallel chains in native ramie cellulose. The one serious problem yet to be resolved concerns the mechanism of conversion of cellulose I into cellulose I1 during mercerization. As the cellulose really is not solubilized during the treatment, it is difficult to envisage a mechanism whereby parallel chains can be converted into an antiparallel orientation. Sarko and O k a n ~ proposed *~ a mechanism whereby this might be possible, but details of this conversion are still poorly understood. Because the X-ray patterns of the native celluloses from all organisms examined are quite similar, it now appears that the weight of the present evidence supports the concept that the chains of all native celluloses are in the parallel orientation. This conclusion has very important implications for the mechanism of polymerization and crystallization of native cellulose. If all the chains are oriented in the same direction, and growth proceeds from one end of the microfibril, only one mechanism of polymerization (either addition at the reducing or nonreducing end) need be invoked. If, on the other hand, the chains are antiparallel, some more-complex kind of mechanism must be envisaged that would allow some chains to be elongated from the reducing end, and others from the nonreducing end. For those who study biosynthesis, it is therefore a relief to learn that the weight of evidence favors the parallel model! Another structural consideration relevant to biosynthesis is the fact that, within a chain, each D-glucosyl residue is rotated approximately 180" with respect to its neighbor, as depicted in 1. Thus, cellobiose may actually be considered to be the true repeating-unit within the chain, and it is necessary to consider whether some activated form of the disaccharide (rather than of D-glucose) might be the donor for chain elongation; this point will be considered again in Section V,2. Finally, the question of the average degree of polymerization (d.p.) of the glucan chains must be considered. Determinations of the d.p.of polysaccharides are usually made by viscosity measurements, but native cellulose, being highly insoluble and often in close association with other cell-wall polysaccharides and lignin, presents special prob(48) .4. J . Stipanovic and A. Sarko, Polymer, 19 (1978)3-8. (49) A. Sarko and T. Okano, Proc. Ekmun-Days Int. Symp. Wood Pulp. Chem., 4 (1981)

91-95.

BIOSYNTHESIS O F CELLULOSE

115

TABLEI Average Degree of Polymerization (d.p.)of Native Celluloses d.p. of cellulose Material

Acetobacter ~ y l i n u m ~ ' * ~ * Gossypum hirsutum (cotton fiber)% Primary cell-wall Secondary cell-wall Acanthamoeba castellanis V~lonia~~ Seed hairsa;" Bast Wood from angiosperms";" Wood from g y r n n o ~ p e r m s " ~ ~ ~ ~~

2,000-3,700;5,700 2,000-6,000 13,000-14,000 2,000-6,000 26,500 10,350 9,550 8,200 8,450

~~

Average of all species examined.

lems, as discussed by Goring and Timell.50Purification of the cellulose by sequential extractions may lead to chain breakage, as may some of the derivatization techniques used for solubilizing the cellulose for viscosity measurements. Thus, the values presented in Table I should be considered to be minimum estimates; however, care has been taken only to cite values obtained by techniques that should lead of the A. xylinum to minimal degradation. A determination of the 6. cellulose by Takai and coworkers51yielded a value of 5,700 residues per chain; values obtained by Man-Figini and Pion52were somewhat lower (2,OOO-3,700). The cellulose of primary cell-walls of higher plants, a classic example being the elongating cotton-fiber,%also has a relatively low and heterogeneous d.p.value ranging from 2,000-6,000, as does the cellulose from A ~ a n t h a m o e b aOn . ~ ~the other hand, the native cellulose of the unicellular alga Vdonia has been to have a d.p.of 26,500, with some molecular-weight species approaching 44,000.The d.p.of the secondary-wall cellulose of higher plants is also much higher than that found in the primary walls. M a ~ x - F i g i n i ~ ~ found a d.p.of 13,OOO-14,000 for the secondary-wall cellulose of the cotton fiber, and the homogeneity of molecular weight was surprisingly high. Goring and Tirnell5O examined other, mature seed-hairs and bast fibers, and also found d.p. values ranging from 7,000 to 15,000.The native cellulose of wood and bark tissues showed values (50)D.A. I. Goring and T. E. Timell, Tappi, 45 (1962)454-460. (51)M.Takai, Y. Tsuta, and S. Watanabe, Polym. ]., 7 (1975)137-146. (52)M. Man-Figini and B. G. Pion, Biochim. Biophys. Acta, 338 (1974)382-393. (53) M. Man-Figini,]. Polym. Sci., Part C, 28 (1969)57-67. (54)W. E. Blanton and C. L. Villemez,]. Protozoal., 25 (1978)264-267. (55)A. Palma, G. Biildt, and S. M. Jovanovic,Makromol. Chem., 177 (1976)1063-1072.

116

DEBORAH P. DELMER

from 7,000 to 10,0o0, the softwood celluloses having, on the average, slightly lower values than those of the h a r d ~ o o d s . ~ ' The length of microfibrils observed far exceeds the length of even the longest of the D-glucan chains. Thus, it would appear that chains are terminated and re-initiated many times during the course of synthesis of a microfibril. The presence of such ends within the microfibril has been proposed by MiilethaleP as being one cause of the amorphous regions in the microfibril. Unfortunately, we have absolutely no idea as to what regulates chain initiation or termination during cellulose biosynthesis. The surprisingly monodisperse, molecular-weight distribution observed by M a ~ x - F i g i n ifor ~ ~ cotton-fiber, secondary-wall cellulose, and the fact that this distribution was not affected by changes in the rate of synthesis, led her to propose that some kind of template mechanism may govern the chain length. However, it is difficult to envisage such a mechanism for polysaccharide synthesis, and no experimental evidence exists to support the hypothesis. Maclachlanj proposed that cellulases may continually break chains and create new initiation-sites, but direct experimental evidence for this is also lacking. An alternative possibility is that imperfect alignment of the D-glucan chains during polymerization and subsequent crystallization could eventually lead, at the region of an active site, to a strain that could lead to termination. As the cellulose of higher-plant and algal secondary-walls is more highly ordered, the strain may develop less frequently, and thus lead to a higher this is, however, at present pure conjecture.

6.;

Iv. CYTOLOGICAL INVESTIGATIONS OF CELLULOSE BIOSYNTHESIS

1. The Site of Cellulose Biosynthesis In the past few years, some impressive observations have been made by microscopists studying cells actively engaged in cellulose synthesis. It is now generally accepted by most workers that, in the bacterium A . xylinum, in most algae, and in all of the higher plants, cellulose is synthesized at the cell surface by an enzyme system localized in the plasma membrane. The notable exception to this conclusion concerns those algae which synthesize a cell wall composed of cellulosic scales; such scales are synthesized intracellularly by way of the Golgi apparatus (see Ref. 57 and references cited therein). a. Observations with Acetobacter xylinum. -In the bacterium A . xyZinum, microscope observations of single cells by Brown and cowork(56) K. Miilethaler,J. Polym. Sci., Purt C, 28 (1969)305-316. (57) R. M . Brown, Jr., and D. K. Romanovicz,Appl. Polym. Symp., 28 (1976) 537-585.

BIOSYNTHESIS OF CELLULOSE

117

and ZaaP clearly showed the synthesis of a ribbon of cellulose, composed of microfibrils, that is attached parallel to the longitudinal axis of the cell. The microfibrils of the ribbon are extruded from a linear row of pores along one side of the cell, and associate to form the ribbon (see Fig. 1).A. xylinum, possessing no flagellae, is considered to be a nonmotile organism but, in a fascinating, motion picture photographed by Brown and discussed in Ref. 59, the bacterium is clearly propelled forward by the force created in synthesizing the rigid ribbon. In a model presented by Brown,Gothe enzymes responsible for polymerization of the D-glucan chains are presumed to exist below the extrusion pores in the lipopolysaccharide layer of the bacterium, but, to date, no conclusive evidence exists for their precise location; it seems equally probable that they could be located in the inner plasma-membrane. As techniques exist for separating the inner and outer membranes of Gram-negative bacteria,6l and as synthesis of (1+4)-P-~-glucan from UDP-glucose can be demonstrated in vitro with cell-free preparations ofA. x y Z i n ~ m ,the ~ * localization ~~ of the enzyme(s) could be attempted. Early attempts by Cooper and M a n l e ~ ~ ~ to perform such experiments were inconclusive. Haigler and coworkersMalso made the interesting observation that polymerization and crystallization of cellulose microfibrils can be “uncoupled.” This was achieved by incubating A. xylinum cells engaged in cellulose synthesis in the presence of the fluorescent brightener Calcofluor White ST, a compound capable of forming hydrogen bonds with free hydroxyl groups. When present during polymerization, it presumably binds to the newly polymerized, D-glucose residues, and thus prevents microfibril assembly. Hence, they observed production of ordered, but not crystalline, material attached perpendicular to the cell axis in place of the normal ribbons. This material was characterized as non-crystalline cellulose by Benziman and coworkers,G5who also made the interesting observation that the rate of polymerization of D-glucose is enhanced up to 4-fold in the presence of Calcofluor White, implying that crystallization may be the rate-limiting step in cellulose biosynthesis. As the microfibrils produced are, normally, rigid structures, it seems possible that the rate of elongation (58) K. Zaar,J. Cell Biol., 80 (1979) 773-777.

(59) R. M. Brown, Jr.,Proc. Ekman-Days Int. Symp. Wood Pulp. Chem., 3 (1981)3-15. (60) R. M. Brown, Jr., Proc. Philip Morris Sci. Symp., 3rd., (1979) 52-123. (61) M. J. Osbom and R. Munson, Methods Enzymol., 31 (1974) 642-653. (62) L. Glaser,J. Biol. Chem., 232 (1958)627-636. (63) D. Cooper and R. S. J. Manley, Biochim. Biophys. Actu, 381 (1975)97-108. (64) C. Haigler, R. M. Brown, Jr., and M. Benziman, Science, 210 (1980)903-906. (65) M. Benziman, C. H. Haigler, R. M. Brown, Jr., A. R. White, and K. M. Cooper, Proc. Natl. Acad. Sci. USA, 77 (1980) 6678-6682.

118

DEBORAH P. DELMER

of an entire microfibril could be constrained by the slowest, individual, catalytic site; when crystalline association between the chains is prevented, such a restriction would be relieved. The rate of cellulose synthesis in A . xylinum has also been shown by Ben-Hayim and Ohadfi6to be stimulated (but only by -30%) by the presence of soluble 0-(carboxymethy1)cellulose (CMC). Haigler and Benziman4I discussed and interpreted these results, and suggested that the high-molecular-weight CMC interferes with synthesis at a higher level of organization, that is, by preventing association of the 3.5-nm fibrils into larger bundles. At this point, it must be mentioned that R. Colvin, who has produced numerous publications on cellulose synthesis in A . xylinum, strongly disagrees with the model of cellulose synthesis proposed by Brown's group and supported by Zaar. Colvin's arguments were presented in a review.' He apparently believes that enzyme(s) localized within the cell produce a soluble precursor (see also, Section V,3) (to cellulose) that is secreted and somehow converted, outside the cell, into crystalline cellulose without the aid of cell-associated enzymes. He thus does not believe that polymerization and crystallization are coupled events that are directed by an enzyme complex within the sudace membranes. There is no question that some evidence now exists for a possible, soluble, high-molecular-weight precursor to cellulose; this topic will be discussed in more detail in Section V,3. Colvin's groiqF?'j*claimed to have observed such an intermediate by electron-microscope observation; however, Willison and coworkers69 argued that these could be artifacts of radiation damage or improper focusing of specimens during microscopy. There is also some reason to question most of Colvin's criticisms of the cytological studies of Brown's group. The images of multiple sites of attachment' of a growing ribbon of cellulose were clearly demonstrated, not only by Brown's group but also by Zaar.jHColvin's techniques of microscopy might not favor the preservation of such structures in intact cells, and certainly not in the membrane preparations that he used for in vitro studies.'" One of his criticisms is, however, partially valid. In the initial study reported by Brown and coworkers,35no carbon source was supplied to the cells before the visual observations were recorded, (66)G. Ben-Ha!-im and I. Ohad,]. Cell Biol., 25 (1965) 191-207. (67) J. H . Colviri and G. G. Leppard, Cun. J . Microhiol., 23 (1977) 701-709. (68)J. R. Colvin, L. L. Sowden, and G. G. Leppard, Cun. J . Microhiol., 23 (1977) 790797. (69)J . H. M . Willison, R. M. Brown, Jr., and S. C. Mueller,]. Microsc. (Oxford), 118 11980) 177-186. (70) J. H. Colvin, PEontu, 149 (1980) 97-107.

BIOSYNTHESIS OF CELLULOSE

119

and thus, Colvin argued7that no cellulose could have been produced under these conditions. However, the observations made by Zaar,58 were conand all subsequent ones by Brown and coworkers,59~60~64~65 ducted in the presence Of D-glUCOSe; it seems probable that sufficient, residual mglucose from the growth medium remained associated with the cells described in Brown’s earlier report.70a b. Observations with Algae and Higher Plants.-Several unicellular algae have served as excellent models for cytological studies of cellulose biosynthesis. These algae deposit a layered, cellulosic cell-wall having a highly ordered pattern of deposition of large microfibrils whose direction of orientation alternates with each successive layer deposited. The older cytological studies with these algae have been well reviewed by R ~ b i n s o nSubsequent .~ research has effectively employed the technique of freeze-fracturing the plasma membrane of these algae, in order to look for structures that could constitute the sites of cellulose synthesis. A brief, but succinct, review of these freeze-fracture studies has been presented by Lloyd.” In one of the first examples of such studies, Robinson and Preston72observed ordered arrays of protein subunits (termed “granule bands”) embedded in the plasma membrane of the alga Oocystis. They suggested that these ordered arrays could be the multi-subunit, cellulose synthetase complexes originally proposed to exist by Preston40in his “orderedgranule hypothesis” of 1964. However, subsequent work by Brown and M o n t e ~ i n o sshowed ~ ~ , ~ ~that the structures observed by Robinson and Preston72were situated on the inner face of the cytoplasmic half (P-face) of the plasma membrane. Brown and Montezinos also observed, in addition to these P-face granule-bands, another type of ordered protein-complex on the inner side of the outer half (E-face) of the plasma membrane. These ordered arrays were nearly always observed to be situated at the ends of microfibrils. They called these ordered arrays of protein subunits “terminal complexes,” and proposed that they constituted the cellulose-synthesizing, enzyme complexes. Because of their precise orientation, it was proposed that the granule bands situated on the opposite face of the plasma membrane play some role in the orientation of the microfibrils. Fig. 2A shows a model of these various complexes as interpreted by Montezinos and Brown.74 (70a) M. Benziman, personal communication. (71) C. Lloyd, Nature, 284 (1980) 596-597. (72) D. G . Robinson and R. D. Preston, Planta, 104 (1972)234-246. (73) R. M. Brown, Jr., and D. Montezinos, Proc. Natl. Acad. Sci. USA, 73 (1976) 143147. (74) D. Montezinos and R. M. Brown, Jr.,J. Suprarnol. Struct., 5 (1976)277-290.

120

DEBORAH P. DELMER

A

B Rosette

I

.

@

7

lOnm 15nm 2Onm

FIG.2.-A. A Model Depicting the Molecular Architecture of the Plasma Membrane of Oocystis apiculata During Secondary-wall F~rmation.'~ [MF, Cellulosic microfibrils; TC, terminal complexes; PTC, paired, terminal complexes; CR?, regions of possible, transmembrane control; GB, granule bands;TC1, impressions of terminal, complex particles; IP, intramembranous particles, AL, region of membrane phospholipids af-

BIOSYNTHESIS OF CELLULOSE

121

Similar, and equally elegant, results have been reported by Giddings and for the alga Microsterias denticulata. This group also observed, by freeze-fracture techniques, the so-called “terminal complexes” associated with the ends of microfibrils. These terminal complexes, however, show some morphological differences from those observed in Oocystis (compare the model of Brown and Montezinos, in Fig. 2A, with that of Giddings and coworkers in Fig. 2B). In Microsterias, the complex consists primarily of a hexagonal array of from 3 to 175 rosettes, consisting of 6 particles each, which fractures with the P-face of the membrane. On the E-face of complementary fractures, particles complementary to the central hole of the P-face rosettes were sometimes evident. No structures similar to the granule bands of Oocystis were apparently observed in Microsterias. The studies on Microsterias may be relevant to an older study by Kiermayer and D ~ b b e r s t e i n who , ~ ~ observed, in thin sections of Microsterias cells, flat vesicles containing regular arrays of 20-nm particles; they suggested that these vesicles are Golgi-derived, and ultimately fuse with the plasma membrane, and thus could constitute precursors to the terminal complexes observed by Giddings and coworker^.^^ Less distinctive, cylindrical, terminal complexes have also been reported in another alga, namely, G l a ~ c o c y s t i s . ~ ~ Terminal complexes have also been claimed to have been observed in freeze-fracture studies of higher-plant cells. However, at least to the inexperienced eye of this biochemist, the images seen are far less clear than those observed with the algae. Thus far, such complexes have been reported in cells of corn root^,^*,^^ radish roots,8O and cotton (75) T. H. Giddings, Jr., D. L. Brown, and L. A. Staehelin,J . Cell Biol., 84 (1980) 327339. (76) 0.Kiermayer and B. Dobberstein, Protoplasma, 77 (1973) 437-451. (77) J. H. M. Willison and R. M.Brown, Jr.,J. Cell Biol., 77 (1978) 103-119. (78) S. C. Mueller, R. M. Brown, Jr., and T. K. Scott,Science, 194 (1976) 949-951. (79) S. C. Mueller and R. M.Brown, Jr.,J. Cell Biol., 84 (1980) 315-326. (80) J. H. M. Willison and B. W. W. Grout, Planta, 140 (1978)57-58. fected by granule bands; and UAL, regions of membrane phospholipids not affected by granule bands.] B. A Model of Cellulose-Fibril Deposition During Secondary-wall Formation in Mic r o s t e r i ~ s[Each . ~ ~ rosette is believed to form one 5-nm microfibril. A row of rosettes forms a set of 5-nm microfibrils which aggregate laterally to form the larger fibrils of the secondary wall. Above: side view. The stippled area in the center of a rosette represents the presumptive site of microfibril formation, although details of its structure, composition, and enzymic activity remain unclear. Below: surface view, with expanded, crosssectional view of cellulose fibrils.]

122

DEBORAH P. DELMER

fibers.x’ In general, the “complexes” in plant cells are smaller, and less distinct, than in the algae, consisting usually of one globule at the end of a microfibril, although Mueller and Brown79also claimed to have observed rosettes similar to those found in Microsterius. It is, perhaps, unfair to criticize too strongly the lack of clarity in these freeze-fracture images. The microfibrils of higher-plant cells are considerably smaller than those found in the algae; thus, the size and complexity of such a plant terminal-complex might be expected to be less than those for the algal cells. It is necessary to mention a report by Grout,H2strongly supported by Colvin,’ which proposed that no such terminal complexes exist in plant cells, but rather that cellulose synthesis proceeds by a relatively slow accumulation of cellulose precursors in the outer regions of the plasma membrane, followed by swift, spontaneous precipitation, and crystallization without the aid of enzymes. Grout’s conclusions were based on deep-etch studies of the outer surface of the plasma membrane of tobacco protoplasts engaged in cell-wall regeneration. He saw no terminal complexes, and only saw microfibrils after a lag of 16 hours after wall removal. However, the complexes, if embedded in the membrane, may only be made visible by a fracturing of the two membrane faces. Furthermore, Klein and D e l m e P observed microfibril deposition in soybean protoplasts within minutes after wall removal, and such microfibrils increase in size progressively with time; thus, there seems no need to invoke a sudden crystallization following a long lag. What is the evidence that the terminal complexes observed are really cellulose-synthesizing complexes? Obviously, cytological procedures do not allow the investigator to “see” the process ofpolymerization; thus, all of the evidence available is circumstantial. Evidence in favor of such a role for the terminal complexes is, in my opinion, strongest for the algae Oocystis and Microsterias. There is no doubt that these complexes are found embedded within the plasma membranes at the ends of microfibrils. In Microsterias, the distance between rows of rosettes is equal to the center-to-center distance between parallel fibrils in the wall. Furthermore, the widest fibrils (those in the center o f a band, see Fig. 2b) are found associated with the longest rows ofrosettes, whereas shorter rows of rosettes appear to give rise to narrower fibrils. In Oocystis, the number of particles per complex has been estimated to approximate the number of D-glucan chains per microfibril. The complexes and associated microfibrils are (81) J . H. IM. Willison and R. M . Brown, Jr., Protoplasma, 92 (1977)21-41. (82) B. W. W. Grout, Plonta, 123 (1975)275-282.

BIOSYNTHESIS OF CELLULOSE

123

always associated with the innermost, developing layer of the wall, and are perpendicular to the previous layer. Montezinos and BrownB3 found that treatment of Oocystis cells with the divalent-cation chelator (ethylenedinitri1o)tetraacetic acid (EDTA) results in dissociation of the complexes from the microfibrils, and the effect is, at least partially, reversible by Mg2+.Such concentrations in this chelator have been shown by other worker^'^^^*^^^ to inhibit cellulose synthesis in vivo. The possible arguments against a role for the terminal complexes are: ( a ) the technical limitations, which do not allow a correlation of the cytology with direct biochemical measurements of cellulose synthesis, and (b) the possibility that the complexes are artifacts resulting from an unnatural aggregation of plasma-membrane proteins during preparation of the samples. Cytologists counter the latter argument with the facts that ( a ) the images are precise and quite reproducible, (b)the cells undergo little or no previous fixation prior to freezing, and (c) the existence of microfibril impressions indicates that the cells were turgid before freezing. In the present author’s opinion, the evidence at present tends to favor the conclusion that the terminal complexes observed, at least in the algae, do represent the sites of cellulose synthesis.

2. Orientation of Microfibrils The cellulosic microfibrils in the cell walls of algae and higher plants often have a characteristic pattern of orientation. In the thin, primary-cell walls of dividing, non-differentiated, higher-plant cells, the orientation of microfibrils tends to be random. However, as such cells begin to elongate, the innermost, newly formed microfibrils tend to become parallel in a direction transverse to the growth axis. According to the “multinet-growth hypothesis” of Roelofsen and Houwink,S6 the realignment of the microfibrils during elongation occurs passively by the forces generated during elongation, although studies by Roland and coworkerP indicated that such a hypothesis may be a somewhat oversimplified view of the orientation of cell-wall components in elongating cells. However, the most striking cases of precise orientation of microfibrils occur in the secondary walls of plant cells, and in the algae. In these instances, the walls consist of distinct layers of pre(83) D. Montezinos and R. M. Brown, Jr.,Cytobios, 23 (1979) 119-139. (84) H. Quader and D. G . Robinson, Eur. I. Cell Biol., 20 (1979)51-56. (85) D. Montezinos and D. P. Delmer, Planta, 148 (1980)305-311. (86) P. A. Roelofsen and A. L. Houwink, Acta Bot. Need., 2 (1953)218-225. (87) J. C. Roland, B. Vian, and D. Reis, Protoplasma, 91 (1977) 125-141.

124

DEBORAH P. DELMER

cisely parallel microfibrils, the directions of which alternate in each succeeding layer. Such cells are not elongating, and some mechanism other than passive forces must be invoked in order to account for the precise orientations observed. Because the bacterium A. xylinum extrudes its newly synthesized cellulose as a ribbon into the growth medium, no mechanism is required for orientation of these microfibrils. However, the observation by Brown, in his motion pictures of living A. xylinum cells, that the cells are propelled forward by the elongation of a relatively rigid, cellulosic ribbon, may be relevant to our understanding of the mechanism of orientation of microfibrils in the maturing walls of algae and higher plants. As the synthesizing complex is embedded in a fluidmosaic membrane, it seems probable that the propelling force created by the act of polymerization could move the complex within the membrane and, therefore, that no external source of energy for such movement need be invoked. (If the complexes do move within the membrane in algal and plant cells, it is worth noting that this constitutes a notable difference from the stationary complexes of A . xylinum. ) The question of interest, therefore, is not how the complex in algae and plants moves, but rather, what determines the precise direction of the motion, such that highly ordered patterns of orientation result. Ever since the discovery, by Ledbetter and Porter,"* of microtubules below the surface of the plasma membrane, suggestions have been made that these structures play some role in microfibril orientation. The suggestion arose because of two observations: that (1) the orientation of microtubules has very frequently, but not always, been observed to be parallel to the orientation of the microfibrils most recently synthesized, and (2) agents, such as colchicine, that disrupt microtubules interfere with the orientation, but not the synthesis, of cellulose microfibrils. The literature pertaining to these studies has been well reviewed by Robinson: Schnepf and cow0rkers,8~Hepler and Palevitz,goand Heath?' In sum, the present evidence seems to favor some role for microtubules in orientation; in some cases, such as the studies on guard cells by Palevitz and H e ~ l e rand , ~ ~a series of papers ~ ~ case ~ " ~ for ~ microon Oocystis by Robinson and ~ o ~ o r k e r s , Rthe (88) M. C. Ledbetter and K. R. Porter,]. Cell Biol., 19 (1963)239-250.

(89) E. Schnepf, G. Roderer, and W. Herth, Planta, 125 (1975)45-62. (90)P. K. Hepler and B. A. Palevitz, Annu. Reti. Plant Physiol., 25 (1974)309-362. (91) I. B. Heath,]. Theor. Biol., 48 (1974)445449. (92)B. A. Palevitz and P. K. Hepler, Planta, 132 (1976)71-93. (93) D. G. Robinson and W. Henog, Cytohiologie, 15 (1977)463-474. (94)H. Quader, 1. Wagenbreth, and D. C. Robinson, CytobioZogie, 18 (1978)39-51. (95) D. G . Robinson and H. Quader, Eur. ]. Cell Biol., 21 (1980)229-230.

BIOSYNTHESIS OF CELLULOSE

125

tubule involvement would seem to be unequivocal, but many questions remain unanswered. It has been difficult to visualize any connection between these microtubules and structures in the plasma membrane, although a peculiar, nonstaining region surrounding the microtubules, first noted by Behnke,% implies the existence of some connection. Thus, the question of the mechanism by which microtubules interact and direct the movement of the synthetase complex remains unanswered. Also unanswered is whether the granule bands observed in Oocystis play some role in orientation and, if so, what their relationship to microtubules may be.

v. THE

MECHANISMOF POLYMERIZATION

The cytological investigations on cellulose biosynthesis already described support the concept that the overall process of cellulose biosynthesis, that is, polymerization of (l-A)-p-D-glucan chains, and the accompanying organization, crystallization, and orientation of microfibrils, is a complex operation that most probably requires a high degree of structural organization. The need to maintain such organization may explain the relatively slow progress achieved to date by workers attempting in vitro synthesis of cellulose by using cell-free preparations. Because convincing in vitro synthesis of true, microfibrillar cellulose has been difficult, if not impossible, to achieve in so many systems, extensive controversy has existed, even for the straightforward question of what activated form(s)of &glucose serve(s) as precursor to cellulose. The following three sub-sections of this article, therefore, deal with this important question. Discussion will be focused primarily on work using cell-free preparations, but will also consider the results of pertinent experiments designed to trace the path of carbon into cellulose in vivo.

1. Involvement of Glycosyl Esters of Nucleoside Diphosphates The most common donors of activated glycosyl groups for polysaccharide biosynthesis have been found to be glycosyl esters of nucleoside diphosphates, and, therefore, it is no surprise that workers in the field have assumed that a D-glucosyl ester of a nucleoside diphosphate is a probable precursor to cellulose. The two compounds that emerged from early studies as the most likely candidates are GDP-glucose and UDP-glucose. To the best of the author’s knowledge, no other Bglucosy1 ester of a nucleoside diphosphate has been successfully used for in vitro synthesis of (1+4)-p-&glucan. (96)0. Behnke, Cytobiologie, 11 (1975)366-381.

126

DEBORAH P. DELMER

A possible role for GDP-glucose emerged from studies by Hassid's group,gi~"x using membrane preparations derived primarily from etiolated hypocotyls of mung beans; they found that GDP-glucose, and no other nucleoside (D-glucosyl diphosphate), could serve as a precursor to (l-+$)-p-D-glucan.Since then, a number of reports on in uitro synthesis of (1+4)-@-~-g1~can from GDP-glucose, using plant extracts, have appeared in the literature; previous review^^,"^ documented these earlier studies, and also discussed some of the problems of interpretation of the results. To summarize these discussions, a number of observations have led to questioning whether this enzyme reaction is really involved at all in cellulose biosynthesis; these are as follows. ( ( 1 ) The level of GDP-glucose in most plants seems to be vanishingly small; for example, in an analysis by Carpita and DelmerZ3of quantities and patterns of labeling of glycosyl esters of nucleoside diphosphates in the developing cotton fiber, no significant level of GDP-glucose was detected by chemical analyses, nor was any radioactivity found in any compound resembling GDP-glucose during labeling experiments in c k ~This . is in marked contrast to the situation for UDP-glucose, where a substantial pool is observed in cotton fibers'L3.YYand other plant tissues.'"0-104In the cotton fiber, the UDPgliicose pool is readily labeled, and shows rapid turnover during pulse-chase experiments.'" These observations were complemented by measurement of'the levels of activity of enzymes responsible for the synthesis of GDP-glucose and UDP-glucose in plants. Only two report^'^^*'^^ exist that document an activity for GDP-glucose pyrophosphorylase (EC 2.7.7.34)in plants (pea seedlings only). In the present author's laboratory, this enzyme has never been demonstrable in any other plant extract examined, and conversations with colleagues in the field, as well as a report by Heiniger and F r a n ~ ,indicated '~~ that similar results have been obtained in a variety of laboratories. In contrast, the activity of UDP-glucose pyrophosphorylase (EC 2.7.7.9) is quite high in all plant tissues examined,"Jo5as are the levels of sucrose synthetasej (EC 2.4.1.13), another enzyme capable of synthesizing (97) C . A. Barber, A. D. Elhein, and W. Z . Hassid,J. B i d . Chem., 239 (1964) 40564061. (93)A. 1).Elbein, C . A. Barber, and W. Z. Hassid,J. Am. Chem. Soc., 86 (1964) 3093 10. (991 G. Franz, Phybochemistry, 8 (1969) 737-741. (100) M. A. Elnaghy sand P. Nordin,Arch. Biochem. Biophys., 113 (1966)72-76. (101) F.A. Isherwood and R. R. Selvendran, Phytochemistry, 9 (1970) 2265-2269. (102) M . M. Smith and B. A. Stone, Biochim. Biophys. Acta, 313 (1973)72-94. (103) G. A. Barber and W. Z. Hassid, Biochim. Biophys. Acta, 86 (1964) 397-399. (104) C. Peaud-Lenoel and M. Axelos, Eur. 1. Biochem., 4 (1968)561-567. (105) U . Heiniger and G . Franz, Plant Sci. Lett., 17 (1980)443-450.

BIOSYNTHESIS OF CELLULOSE

127

UDP-glucose. It might be suggested that, although pool levels of GDP-glucose are low, the flux through the pool could be high; however, observation of a high capacity for synthesis of GDP-glucose would be expected were this so. (b) The incorporation of radioactivity from GDP-glucose into D-glUcan is linear with time for only a few minutes, and is often stimulated by the addition of unlabeled G D P - m a n n o ~ e . ~ , 'Under ~~-~~ such ~ circumstances, glucomannan, a linear heteropolymer containing p-( 1 4 ) linkages of both glucose and mannose, is produced. V i l l e m e ~ ' ~ ~ J ~ * therefore suggested that the GDP-glucose:(ld)-p-D-glucan synthetase (EC 2.4.1.29)'09 really functions in vivo in the synthesis of the glucomannan. This is a quite plausible explanation; it is puzzling, however, that glucomannans are not common constituents of the primary cell-walls of plants, and most of the tissues wherein this enzyme was observed contain cells having such primary walls. In this regard, it is also of interest that Delmer and coworkers1l0showed that, in the developing cotton-fiber, the GDP-glucose:(ld)-P-D-glucan synthetase is active only during the period of fiber elongation and primary-wall synthesis, and activity declines abruptly with the onset of massive, secondary-wall synthesis of cellulose. Thus, the case against a role for GDP-glucose as a precursor to cellulose seemed to have been established. However, the idea has been resurrected by the studies of Hopp and coworkers," who studied in vitro synthesis of cellulose using membrane preparations derived from the alga Prototheca zopfii. They observed that a combination of UDP-glucose and GDP-glucose was required for synthesis of alkaliinsoluble (14)-p-D-glUCan. This unusual finding has not been observed in our laboratory using plant extracts,"' nor reported elsewhere in the literature, and it remains to be seen whether the observation in Prototheca has general significance. It should also be noted that the characterization of the cellulosic product by Hopp and cow o r k e r ~was ~ ~ not extensive. A considerably stronger case can be made for a role for UDP-glucose as precursor to cellulose. The first convincing report of in vitro synthesis of alkali-insoluble (1+4)-p-Dglucan from UDP-glucose (106) A. D. Elbein,]. Biol. Chem., 244 (1969) 1608-1616. (107) C. L. Villemez, Biochem. I., 121 (1971) 151-157. (108) C. L. Villemez and J. S. Heller, Nature, 227 (1970)80-81. (109) Previously, this enzyme and the one that utilizes UDP-glucose have been designated as cellulose synthetases (EC 2.4.1.29 and EC 2.4.1.12, respectively). These designations should still be considered questionable.

(110) D. P. Delmer, C. A. Beasley, and L. Ordin, Plant Physiol., 53 (1974) 149-154. (111) D. P. Delmer, unpublished results.

128

DEBORAH P. DELMER

came from Glaser's studieP using membrane preparations derived from Acetobacter xylinum. This observation has been repeated nu. ~ , GDP-glucose ~~~~ pyromerous times by others with A . x y l i n ~ m The phosphorylase has not been found in A . xylinum, nor does GDP-glucase serve as a substrate for the synthesis of D-glucan in vitro.1° Careful analyses, by Swissa and coworkers: of the flux of carbon through the UDP-glucose pool in uivo in A. xylinum also fully supported a role for UDP-glucose as precursor. However, some caution is in order, as Sanderman and D e k k e F reported that, under the conditions they employed, UDP-glucose served in uitro as a precursor to a water-soluble ( l + Z ) - ~ - ~ g l u c ainn A. xylinum. This finding has been confi~med,"~ but it was also found that, in addition to the water-soluble (l+Z)-p-D-glucan, alkali-soluble and -insoluble (l+i)-P-D-glUcans were also produced from UDP-glucose in uitro. In the slime and A c a n t h a m e b ~ , 'studies ~ in vitro supported a role for UDP-glucose as precursor to (1+4)-P-~glucan,and the reaction products were reasonably well, although not extensively, characterized in these studies. In higher plants, the situation has been greatly complicated by the presence, in almost all plant extracts, of a highly active UDP-glucose:(1+3)-p-~-glucansynthetase (EC 2.4.1.34) which was first demonstrated by Feingold and coworker^"^ in mung-bean seedlings. This enzyme, probably localized in the plasma memb~-ane,"~*"~ displays a relatively low affinity for UDP-glucose (apparent K,,, in the millimolar range), and sigmoidal V uersus S kinetics are often observed, suggesting activation by the substrate, UDP-glucose. The enzyme has often been found to be activated by such p-linked disaccharides as cellobiose and l a m i n a r a b i o ~ e . ~ JThe ~ ~ JDglucan *~ products display a range of solubilities, from water-soluble to alkali-insoluble. The fascinating aspect about this enzyme is its high activity in uitro in extracts derived from tissues that normally contain little or no (1+3)-fi-D-glucan. [Exand cotton fibersY1l8 which conceptions to this are lily pollen-tube~"~ tain active enzyme, and also (1+3)-/3-~glucanas a natural wall-constituent.] In most plants, callose, a (1+3)-p-~-glucan, appears as a wound polymer, and the obvious conclusion is that this enzyme is normally latent in most plant cells and is activated by wounding and also (112) H. Sanderman and R. F. H. Dekker, FEBS Lett., 107 (1979) 237-240. (113) Y. Aloni, M. Benziman, and D. P. Delmer, unpublished results. (114) D. S. Feingold, E. F. Neufeld, and W. Z. Hassid,]. Biol. Chern., 233 (1958)783787. (115) R. L. Anderson and P. M. Ray, Plant Physiol., 61 (1978) 723-730. (116) P. H. Quail, Annu. Rev. Plant Physiol., 30 (1979) 425-484. (117) D. Southworth and D. B. Dickinson, Plant Physiol., 56 (1975)83-87. (118) D. P. Delmer, U. Heiniger, and C. Kulow, Plant Physiol., 59 (1977)713-718.

BIOSYNTHESIS OF CELLULOSE

129

by the process of preparation of cell-free extracts. The mechanism of such possible activation is not yet understood, although activation of a similar enzyme in fungi by protease has been r e p ~ r t e d . ~ ~ ~ J ~ ~ Nevertheless, this enzyme, fascinating though it may be, has created havoc in the field of cellulose biosynthesis, as numerous claims of the synthesis of cellulose from UDP-glucose in uitro have been erroneous, because of poor characterization of the product, when, in fact, (1+3)-p-~-glucanwas the compound actually produced in vitro. (Primary references have been discussed by Delmer.3) Despite this situation, there are some reports of synthesis of ( 1 4 ) - p - ~ glucan from UDP-glucose where the linkage of the reaction product was reasonably well established; the classic example of this is a Golgilocalized UDP-g1ucose:glucan synthetase first demonstrated by Ray and coworkerslZ1in membranes derived from pea tissue. This enzyme can be distinguished from the UDP-glucose:(1+3)-p-~-glucansynthetase by virtue of its much lower K, for UDP-glucose, and its specific requirement for Mgz+.The two different glucan synthetases, found in many plant tissues, have evolved as marker enzymes in plants for plasma membrane [UDP-glucose:(1+3)-p-~glucansynthetase, called glucan synthetase 111 and Golgi membranes [UDP-glucose:(1 4 ) - p - ~ glucan synthetase (EC 2.4.1.12), called glucan synthetase I], although caution in their use has been advised by Quail.l16For example, glucan synthetase I activity has also been reported by Van der Woude and coworkerslZ2to occur in the plasma membranes of onion stem-tissue. A crucial question, for the higher-plant systems especially, is whether such a UDP-glucose:(l4)-/3-D-glucan synthetase is really a cellulose synthetase. First, the usual localization in Golgi-derived membranes is suspect, although it might be argued that this enzyme is nascent cellulose synthetase en route to the plasma membrane; if it were, it should also be expected to be found more commonly in plasma membrane as well. Second, in tissues engaged in primary-wall synthesis, several other cell-wall polymers, such as the xyloglucan common to dicotyledonous, primary cell-walls and the mixed-linkage (1+3, 14)-p-D-glucan of grass cell-walls, also contain P-D( 1 4 ) linked Dglucosyl residues.6 Ray1= extensively characterized a pea UDP-xy1ose:xylosyl transferase, and found that its activity is stimu(119) M. Fevre and M. Rougier, Planta, 151 (1981)232-241. (120) M. C. Wang and S. Bartnicki-Garcia,Arch.Biochem. Biophys., 175 (1976) 351354. (121) P. M. Ray, T. L. Shininger, and M. M. Ray, Proc. Natl. Acad. Sci. USA, 64 (1969) 605-612. (122) W. J. Van der Woude, C. A. Lembi, D. M. Moore, J. I. Kindinger, and L. Ordin, Plant Physiol., 54 (1974)333-340. (123) P. M. Ray, Biochim. Biophys. Acta, 629 (1980)431-444.

DEBORAH P. DELMER

130

TABLEI1 “Effect of Concentration of UDP-xylose on Stimulation by U D P - g l u c o ~ e ~ ~ ~ Incorporation of [‘4Clxylose (pmol) at [12C]-UDP-glucoseconcentration ( p M )

UDP-xylose IPM 1

0

1.5

0.25 1.5 10 50

5.0 17 38 43

8.1 23 51 39

250

8.6 46 239 256

A11 assays contained 1 mM MnC1, and 4.5 nCi of UDP-[’*C]xylose, plus sufficient unlabeled UDP-xylose and UDP-glucose to give the con‘I

centrations indicated.

lated by addition of UDP-glucose (see Table 11). Under these conditions, oligosaccharides having structures resembling fragments of xyloglucan can be prepared from the reaction products. He concluded that the UDP-ghcose:( l-*4)-&D-glUcan synthetase probably plays a role in the synthesis of xyloglucan rather than of cellulose. This enzyme is most probably one of the enzymes studied by Maclachlan’s group,’24who studied the synthesis of cellulose, also by using pea tissue. They originally reported a “cell surface cellulose synthetase” activity in pea epicotyl-slicesfZ4; however, later studies30 revealed that, at high concentrations of UDP-glucose (which were used in the initial studies), (1+3)-P-D-glucan was mostly produced, whereas, at low concentrations of UDP-glucose, an increase in P-D-(1-4) linkages was observed. Similar problems exist in studies with cereal tissues; for example, the careful work of Stone and coworkers’02”25 on ryegrass may be cited; this showed that, in addition to (1+3)-p-D-glucan and (1-4)p-D-glucan, a mixed-linkage Dglucan product can be produced from UDP-glucose, and that the ratio of (1-4) to (1+3) linkages in the total D-giucan products increases as the concentration of UDP-glucose is lowe red. In summary, it seems that convincing in zjitro synthesis of cellulose from UDP-glucose using plant extracts has never been conclusively demonstrated. The reader should note that, even for the case ofA. xylinuni and other lower organisms, the in vitro products have been re(1s)C . Shore, Y. Raymond, and C . A. Maclachlan, Plant Physiol, 56 (1975) 34-40. (125J J . A. Cook, C . B. Fincher, F. Keller, and B. A. Stone, in J. J. Marshall (Ed.),Mechunisms of Sacchoride Polymerimtion and Depolymerizution, Academic Press, New York, 1980, pp. 301-315.

BIOSYNTHESIS OF CELLULOSE

131

ferred to herein as (1+4)-p-D-g1ucan7not as cellulose. In contrast to the synthesis of chitin, where the in vitro, microfibrillar product has been characterized (chemically, by electron microscopy, and by X-ray diffraction) as authentic hiti in,'^^^'^^ this has not been done for in vitro studies with cellulose biosynthesis. Interestingly, cellulose synthesized in vivo is resistant to solubilization by a mixture of concentrated acetic and nitric acids (the so-called “Updegraff reagent”; for examples of its effects on native cellulose, see Refs. 21 and 26). However, noncrystalline cellulose synthesized in Oocystis in the presence of Calcofluor White is s01ubilized’~~~; and, from discussions with colleagues in the field, it appears that all of the (1+4)-P-Dglucans synthesized in vitro are also solubilized by this reagent, a finding that suggests that these products are noncrystalline. Robinson and Preston12*searched without success for an X-ray diffraction diagram characteristic of cellulose I in the in vitro products derived from UDPglucose in mung-bean seedlings. More significantly, C01vin~~ also did not observe such a pattern from the dried, untreated products of incubation ofA .xylinum membranes with UDP-glucose under conditions where (1+4)-P-D-glUCan is known to be produced. Such a result suggests that true, organized microfibrillar cellulose I is not the in vitro product, and, until proof is otherwise forthcoming, the products should be viewed simply as poly-D-glucose chains that may or may not be some nascent form of cellulose. It is of interest, however, that an X-ray pattern having some reflections characteristic of cellulose 11, as well as electron micrographs of apparent microfibrillar material, were obtained by Colvin after treatment of the products with alkali.70It is possible, therefore, that such “mercerization” can lead to the subsequent crystallization of such a “nascent” form of cellulose, but, as the mercerized product is cellulose 11, this mechanism of microfibril formation certainly does not mimic the in vivo mechanism of assembly. A comprehensive study by Carpita and Delme1-,2~ designed to trace the flow of carbon in vivo from D-glucose to cellulose in the developing cotton fiber, offered strong support for the concept that UDPglucose is a precursor to secondary-wall cellulose in this higher-plant cell. Using cotton fibers cultured in vitro and supplied with radioactive Dglucose, pool sizes and rates of accumulation of label into D-glucose phosphates, UDP-glucose, and end products known, or sus(126) J. Ruiz-Herrera, V. 0. Sing, W. J. Van der Woude, and S. Bartnicki-Garcia, Proc. Natl. Acad. Sci. USA, 72 (1975) 2706-2710. (127) J. Ruiz-Herrera and S. Bartnicki-Garcia, Science, 186 (1974) 357-359. (127a) D. G. Robinson, personal communication. (128) D. G. Robinson and R. D. Preston, Biochim. Biophys. Acta, 273 (1972) 336-345.

132

DEBORAH P. DELMER

pected, to be derived horn UDP-glucose were measured. Computer analyses of the data allowed construction of a model of carbon metabolism in the cotton fiber that indicated that the flux of carbon through the UDP-glucose pool was more than sufficient to account for the combined rates of synthesis of sucrose, steryl Dglucosides, (1+3)-p-D-glucan, and cellulose. No other glycosyl ester of a nucleoside diphosphate displayed an appropriate pattern of labeling consistent with precursor properties. Thus, in summary, both in vitro and in vivo studies in A. xylinum, and in vitro studies with other, lower organisms, supported the concept that UDP-glucose serves as a precursor to a (1+4)-/3-D-glucan that is most probably related to cellulose; in higher plants, in vitro evidence for such synthesis, either from GDP-glucose or UDP-glucose, is not yet convincing. However, levels of UDP-glucose, and capacity for synthesis of UDP-glucose, are quite high in plant tissues; and in vivo labeling studies strongly support a role for UDP-glucose as precursor to cellulose in higher plants.

2. Possible Involvement of Lipid Intermediates Major credit should be given to Khan and C ~ l v i n who, ' ~ ~ while studying the synthesis of cellulose in A. xytinum, first evolved the concept of a lipid intermediate in polysaccharide synthesis. Since then, a role for lipid intermediates in cell-wall synthesis in bacteria, and in glycoprotein synthesis in both plants and animals, has been well documented.'80 In these cases, the lipids are phosphorylated polyprenols containing, typically, 11 isoprenoid units (undecaprenol) in the prokaryotes, and 16-20 units (dolichols) in the eukaryotes, including higher plants. In addition, the dolichols are distinguished from undecaprenol by having an a-saturated isoprene unit. Despite the impressive progress made in regard to the part played by such lipid-linked saccharides in the synthesis of complex carbohydrates, a role for such compounds in cellulose synthesis has yet to be firmly established.

a. Acetobacter -hum.-The evidence for such an intermediate in the early work by Colvin's group was indirect, consisting mainly of electron-microscope observation of the appearance of fibrillar

material following incubation of relatively crude ethanol extracts from

(129) A. W. Khan and J. R. Colvin, Science, 133 (1961) 2014-2015. (130) A. D. Elbein,Annu. Reu. Plant Physiol., 30 (1979)239-272.

BIOSYNTHESIS OF CELLULOSE

133

A. xylinum cells with a crude enzyme derived from the growth medium.129J31 Garcia and coworkers,132using EDTA-treated cells, and Kjosbakken and C ~ l v i n , ' using ~ ~ membrane preparations from A. xylinum, demonstrated synthesis, from UDP-glucose, of glycolipids having properties resembling those of polyprenol Dglucosyl phosphate and containing D-glucosyl, mgalactosyl, or cellobiosyl groups. The possibility of the existence of a cellobiosyl-lipid is of special interest, because, as mentioned previously, cellobiose may be considered to be the true repeating-unit in a (1+4)-p-D-glucan. However, for a number of reasons, the significance of the findings with respect to cellulose biosynthesis remains uncertain. First, the glycolipids have never been purified, and conclusively characterized structurally. Second, no evidence for turnover of these lipids or for subsequent incorporation into (1+4)-p-D-glucan has been presented. Third, Benziman's has been unable to reproduce many of the details of the work of Garcia and coworkers.132Fourth, sander man^^'^^ claimed that the disaccharide attached to the lipid is maltose, not cellobiose, and fifth, a report by Couso and indicated that incubation of A. xylinum membranes with UDP-glucose and GDP-mannose results in the synthesis of a prenyl (Dmannosyl-cellobiosyl diphosphate) that most probably plays no role in cellulose synthesis. Also noteworthy, but not conclusive, is the observation that bacitracin, an inhibitor of lipid-linked reactions involved in prokaryotic, cef !-wall synthesis, does not inhibit cellulose synthesis in viuo in A. xyZinwn.10

b. Prototheca zopfii.-Hopp and coworkers17also reported the synthesis, from UDP-glucose, of glycolipids resembling polyprenol (glycosy1 phosphate) by using membranes derived from Prototheca zopfii. The lipids produced were characterized as lipid-P-glucose, lipid-PPglucose, and lipid-PP-oligosaccharidescontaining (1+4)-p-Dglucosyl residues. Evidence was then given that these glycolipids serve as a precursor to a water-soluble polymer that also contains (1+4)-p-D-glu-

(131)A. W.Khan and J. R. Colvin,]. Polym. Sci., 51 (1961)1-9. (132)R. C. Garcia, E. Recondo, and M. A. Dankert,Eur.]. Biochem., 43 (1974)93-105. (133)J. Kjosbakken and J. R. Colvin, in F. Loewus (Ed.),Biogenesis ofPlant Cell Wall Polysaccharides, Academic Press, New York, 1975,pp. 361-371. (133a)M.Benziman, personal communication. (134)H. Sanderman, FEBS Lett., 81 (1977)294-298. (135)R. 0. Couso, L. Ielpi, R. C. Garcia, and M. A. Dankert,Arch. Biochem. Biophys., 204 (1980)434-443.

134

DEBORAH P. DELMER

cosy1 residues. As already mentioned, they then observed, on addition of GDP-glucose, production of small proportions of alkali-insoluble Dglucan that was briefly, but not conclusively, characterized as ( 1 4 ) p-D-glucan. Although the lipids were not purified, their characterization was sufficiently extensive to indicate that they probably did contain single D-glucosyl or (1-+4)-~-~-glucosyl residues. However, the lack of conclusive characterization of the cellulosic product still leaves open the question of the role of these lipids in cellulose synthesis. It is of interest, however, that, in a subsequent report, these workers showed that coumarin, an inhibitor of cellulose biosynthesis (see Section V,4) did inhibit the transfer of oligosaccharides from the lipids to the water-soluble, polymeric rnate1ia1.I~~ c. Higher Plants.-Essentially no direct evidence exists to support or refute, conclusively, a role for lipid intermediates in cellulose biosynthesis in higher plants. Forsee and E1beinls7reported the synthesis from UDP-glucose of low amounts of a compound resembling polyprenol (D-glucosyl phosphate) by using extracts derived from cotton fibers, but gave no evidence for its further participation in cellulose biosynthesis. Similarly, Pont-Lezica and coworkersz3*were able to demonstrate the presence of a lipid acceptor in several plant-tissues that would accept radioactivity from UDP-D-[14C]glucoseto afford a compound resembling dolichol-P-glucose. As dolichol-P-glucose is also known to participate in protein glycosylation of glycoproteins of the high-mannose type,'"* the observation of synthesis of such a compound does not necessarily implicate its role in D-glucan synthesis. In the present author's laboratory, substantial activity for in vitro synthesis of polyprenol (D-glucosyl phosphate) from UDP-glucose or GDPglucose, using plant extracts,'" has never been detected, despite the fact that synthesis of a compound proved by extensive structural analyses to be dolichol-P-mannose is readily demonstrable by using GDPmannose as the substrate.13gFurthermore, exhaustive analyses of the pattern of labeling of lipids in uiuo, in developing cotton-fibers actively engaged in cellulose synthesis, revealed no substantial levels of labeled D-gliicolipids that might serve as candidates for precursors to cellulose.14uSimilar, negative results were obtained in the presence of specific inhibitors of cellulose, namely, 2,6-dichlorobenzonitrile and (136) H. E. Hopp, P. A. Romero, and R. Pont-Lezica, FEBS Lett., 86 (1978) 259-262. (137) W. T. Forsee and A. D. Elbein,]. Biol. Chem., 218 (1973) 2858-2867. (138) H.Pont-Lezica, C. T. Brett, P. R. Martinez, and M. A. Dankert, Biochem. Biophys. Res. Commun., 66 (1975)980-987. (139) D. P. Delmer, C. Kulow, and M. C. Ericson, Plant Physiol., 61 (1978)25-29. (140) D. Montezinos and D. P. Delmer, unpublished res-ults.

BIOSYNTHESIS OF CELLULOSE

135

coumarin (see Section V,4), which were added in the hope of causing accumulation of such precursors. Thus, for higher plants, it may only be concluded that no evidence exists to support a role for such intermediates; if they do exist, they must be present in very low, steadystate levels, and exhibit very rapid turnover, conditions that may have precluded their detection. Knowledge of the direction of chain growth of a polysaccharide is valuable for assessing the mechanism of polymerization. Thus, it is known that “conventional” synthesis of a polysaccharide, involving direct donation of glycosyl groups from glycosyl esters of nucleoside diphosphates, results in growth of the polysaccharide from the nonreducing end of the chain, whereas addition of sugar residues by way of lipid intermediates has been observed to result in growth of the chain from the reducing end.141The synthesis of dextrans utilizing sucrose as the high-energy donor also results in growth from the reducing end of the chain.141Unfortunately, no information is yet available on the direction of growth of cellulose chains, and the high d.p.of these chains is likely to make such analyses exceedingly difficult. 3. Possible Involvement of High-molecular-weight Precursors to Cellulose

In addition to the suggested, but not proved, role for lipid intermediates, much speculation has appeared on the possibility that there may also exist some sort of high-molecular-weight precursor to cellulose. Here again, suggestive evidence exists for such a polymer, but conclusive proof of its existence is still lacking. a. Acetobacter ryZinum.-Early work with A. x y l i n u m by BenHayim and Ohad66gave evidence for a soluble form of “filtrable cellulose.” However, in these experiments, the possibility that some small fibrils of cellulose might have passed through the filters was not rigorously excluded. For many years, Colvin has been an advocate of the .~ existence of a soluble, intermediate polymer in A. x y Z i n ~ r nKjosbakken and C01vin’~~ incubated A. xy h u m membrane-preparations with radioactive UDP-gIucose, and observed that the radioactive product sedimented as a peak having a density substantially lower than that of cellulose. Although they interpreted this result as indicating that a transient D-glucan of lower density was produced, it seems more probable that, based on the density of the sedimented material (-1.20; even lower than that of pure proteins), they had simply observed a (141) J. F. Robyt, Trends Biochem. Sci., 4 (1979)47-49. (142) J. Kjosbakken and J. R. Colvin, Cen.1. Microbial., 21 (1975) 111-120.

136

DEBORAH P. DELMER

continued association of the radioactive products with the membrane vesicles. Such vesicles, prepared by sonic disruption of bacteria, are often inverted; therefore, it is possible that UDP-glucose, when accepted as substrate by the outer (inverted) face of the vesicles, leads to synthesis of polymers that are deposited in the interior of the vesicles. N o evidence for any conversion of the “intermediate” product into cellulose was given. In another report, by King and C ~ l v i n ,a’ radio~~ active, borate-soluble, polymeric fraction was isolated following incubation of A. xylinum membrane-preparations with radioactive UDPglucose. Some evidence was given that the product was a glucan of molecular weight >30,000; when the radioactive material isolated was re-incubated with the membranes, a small fraction of the radioactivity was transferred to the cellulosic fraction. However, as values were given as the percentage of the total radioactivity supplied, it is difficult to assess the statistical significance of this result. Although of some potential interest, further characterization of this borate-soluble material has not yet been reported. In later s t ~ d i e s , ’ Colvin ~ ~ J ~ and ~ coworkers isolated a fraction, insoluble in 60% ethanol but soluble in water, from the medium in which A . zylinum cells had been grown. Methylation analysis indicated that the material contained a glucan having p-(1-+4) linkages with single glucosyl groups as branches at 0-2 of every third glucosyl residue, on the average. They suggested that this water-soluble glucan was a precursor to cellulose, but gave no proof of a precursor-product relationship. Furthermore, the significance of this polymer with respect to the synthesis of cellulose has since been questioned by Colvin and cow o r k e r ~ , ’and ~ ~ the possible relationship between this polymer and the borate-soluble polymer previously studied is also not clear. Benziman and c o w ~ r k e r soffered ~ ~ ’ ~ additional evidence of a different sort for high-molecular-weight precursors to cellulose in A. xylinum. These workers monitored the kinetics of labeling of various cellular fiactions during incubation of “resting cells” (lacking an external source of nitrogen) in radioactive glucose. Following extraction of the labeled cells with chloroform-methanol, they were able to extract labeled, non-dialyzable, water- and alkali-soluble polymeric materials. During a subsequent chase with unlabeled glucose, radioactivity declined in these fractions, and a corresponding increase in radioactivity (143) G. C . S. King and J. R. Colvin, A w l . Polym. Symp., 28 (1976)623-636. (144) J. R. Colvin, L. Chene, L. C. Sowden, and M. Takai, Can.]. Biockm., 55 (1977) 1057- 1063. (145) L. C. Sowden and J. R. Colvin, Can. 1. Microbiol., 24 (1978) 772-777. (146) J. R. Colvin, L. C. Sowden, V. Daoust, and M. B. Perry, Can. J . Biochem., 57 (1979) 1284-1288.

BIOSYNTHESIS OF CELLULOSE

137

Time ( m i d

FIG.3.-Pulse-chase Experiment with Acetobacter ryZinum.1° {Incorporation of D ['4C]glucose (3,300 c.p.m. per nmol) into the water- and alkali-soluble fractions, and its subsequent transfer from these fractions into cellulose. In the pulse, cells were incubated in 3 mM ~ [ ~ ~ C l g l u c at o s0" e in buffer at pH 6.0; at the time of the chase, cells were diluted in cold buffer, centrifuged, and re-incubated at 30" in buffer either conor lacking &glucose (--).} taining 40 mM unlabeled Dglucose (-)

in cellulose was observed (see Fig. 3).The kinetics of chase suggested that the two fractions (water-soluble and alkali-soluble) were distinct, and that the alkali-soluble polymer(s)served as precursor to the watersoluble material which, in turn, served as precursor to cellulose. The fractions were rendered dialyzable by digestion with crude cellulase, and partially dialyzable by treatment with pronase. Thus, the authors suggested that these polymers might be glucoproteins that serve as precursors to cellulose. Subsequent methylation analysis of these fractions indicated that the radioactivity is mainly located in (1+4)-glucosyl residues, with lesser, and variable, proportions of (1-*2)-glucosyl residues.147Thus, although these fractions have not yet been purified, or completely characterized structurally, they seem to possess properties consistent with a role as precursors to cellulose. b. Protothecu zopfiL-Studies by Hopp and coworkers,17described earlier in connection with the alga Prototheca xopfii, indicated that a radioactive, water-soluble polymer is produced when membrane fractions are incubated with radioactive UDP-glucose. As -40% of the polymer was "hydrolyzed" by pronase treatment, the authors sug(147) U. Rothschild, M. Benziman, and D. P. Delmer, unpublished results.

138

DEBORAH P. DELMER

gested that the polymer is a glucoprotein. The existence of p-D-(1+4)glucosyl residues in the polymer was reasonably well established. However, as in the work with A. xylinnm, rigorous proof that the polymer was a glucoprotein is still lacking; and a role for this polymer as preciirsor to cellulose is still not definitely established. The Prototlzecci \ystem does, however, appear to be a promising one for in vitro studies, and it is to be hoped that future work will clarify the nature of this water-soluble polymer.

c. Higher Plants.- Some circumstantial evidence for a high-moleciilar-weight precursor to cellulose was also educed from studies with higher plants. MortimeP" found that radioactive a-cellulose, formed in barley leaves and sugar beet by labeling with radioactive carbon dioxide, contained a distinct fraction of glucan, of higher specific activity, that could be released by extraction of the a-cellulose with hot, dilute trichloroacetic acid. Following a chase with unlabeled CO, , this radioactivity was transferred to the portion of the a-cellulose fraction that was insoluble in this acid. An intermediate role was suggested for this glucan; an alternative interpretation is, however, that the glucosyl residues most recently incorporated were the most accessible to partial hydrolysis with acid, resulting in the release, by trichloroacetic acid, of glucan fragments having low molecular weights. Satoh and coworkers149observed a somewhat comparable phenomenon during i n cioo labeling of mung-bean hypocotyl-segments in radioactive glucose. However, in this instance, a glucan fraction of high specific activity, that could be hydrolyzed by an impure cellulase to yield glucose and (probably) cellobiose, was found associated with membranes and not with the cell-wall fraction, as was the case in Mortimer's study."* Some turnover of the radioactivity in this fraction could be observed during an in oioo, pulse-chase experiment; furthermore, the synthesis ofthe "cytoplasmic" glucan could be inhibited by coumarin, a relatively specific inhibitor of cellulose synthesis (see Section V,4). These studies appear to be of potential importance; nevertheless, definitive, structural characterization of this fraction as (~+4)-~-D-glucan is required, and, as yet, no further analysis of this fraction has been published. In 1975, FranzI5Oreported that incubation of mung-bean membranepreparations with radioactive UDP-glucose resulted in the production of a variety of polymeric products, one of which was identified as a glucoprotein. The radioactivity associated with this presumed glucoprotein could be solubilized by treatment of the alkali-insoluble prod(148)D. C. Mortimer, C a n . / . Bot., 41 (1963) 995-1004. (149) S. Satoh, K. Matsuda, and K. Tamari, Plant Cell Physiol., 17 (1976) 1213-1254. (150) C . Franz, A p p l . Polym. Symp., 28 (1976) 611-621.

BIOSYNTHESIS OF CELLULOSE

139

ucts with pronase, or by hydrazinolysis. The released material contained all of its radioactivity in glucose residues; upon digestion with an impure cellulase, compounds migrating coincident with cellobiose and cellotriose standards, during paper chromatography, were released. Some turnover of the “glucoprotein” fraction was indicated in pulse-chase experiments. These results appeared quite promising, but surprisingly have not been pursued further by Franz. As with the results of Satoh and coworkers,149the characterization of the glucan moiety was not complete; crude cellulases were employed and, in both studies, it is uncertain whether the chromatographic techniques employed really allowed resolution of di-, tri-, and tetra-saccharides having different linkages. Quite a different sort of possible, high-molecular-weight precursor to cellulose, namely, ( 1+3)-p-D-g1ucan7has also been considered for higher plants. This suggestion has arisen primarily from studies with developing cotton-fibers by the groups of Delmer, Meier, and Waterkeyn. Meinert and DelmeP first observed the appearance of substantial quantities of (1+3)-glucosyl residues in cell walls of cotton fibers during the early stages of secondary-wall formation. Subsequently, Maltby and coworkers151and Huwyler and coworker^^^*'^^ definitely characterized this material as (1+3)-/%D-glUCan.Some peculiarities in the pattern of labeling of the (1+3)-p-D-glucan in viuo, using cultured cotton-fibers, led Maltby and coworkers151to consider whether this glucan might exhibit turnover and, perhaps, serve as a precursor to cellulose; thus, they observed that, in short-term, labeling experiments, the rate of incorporation of label into (1+3)-p-D-glucan exceeded that expected on the basis of chemical analyses of levels of accumulation of (1+3)-P-D-glUCan in the cell wall at this stage of development. However, in repeated, pulse-chase experiments, significant turnover of this glucan fraction could not be demonstrated (see Fig. 4A). also observed, during short-term labeling Pillonel and of fibers, using intact plants, that (1+3)-p-D-glucan is synthesized at a rate that appears to exceed the accumulation observed in the walls, and they, also, suggested that turnover must occur. In subsequent studies, Meier and coworker^^^^,^^^ were able to demonstrate a slow turnover of the (1+3)-p-D-glUCan fraction in vivo (see Fig. 4B).Tech(151) D. Maltby, N. C. Carpita, D. Montezinos, and D. P. Delmer, Plant Physiol., 63 (1979) 1158-1164. (152) H. R. Huwyler, G . Franz, and H. Meier, Plant Sci. Lett., 12 (1978) 55-62. (153) C. Pillonel, A. J. Buchala, and H. Meier, Planta, 149 (1980)306-312. (154) H. Meier, L. Buchs, A. J. Buchala, and T. Homewood, Nature, 289 (1981) 821822. (155) H. Meier, in Ref. 27, pp. 75-83.

m-s 3 - A -

a 0

c 0

0

60

rnin

120

Ieo

Days after beginning of 14C02 pulse FIG.4.-A. Pulse-chase Experiment using Cotton Fibers (Gossypium hirsuturn) Cultured in oitro; Kinetics of Labeling of Cellulose and (1+3)-/3-~-Glucan.'~~ {Fibers, with their associated ovules, were incubated in ~-['~C]glucose (20 mM; 0.08 pCi per pmol). At the time of the chase, half of the remaining ovules with fibers were briefly rinsed and then incubated in 100 mM unlabeled r>-glucose. Cellulose and (1+3)-p-~-glucan were analyzed as d e ~ c r i b e d . ' ~Key: ' (-) pulse; (---) chase; (0)cellulose; (0)water-soluble (1+3)-p-~-glucan; (A) water-insoluble (1-+3)-/3-~-glucan.) B. Pulse-chase Feeding1Mof W02 to Branches of Intact Plants of Gossypium arhoreirm L. ['*C02 (7.4 MBq; 2.0 FCi) was fed in the morning in bright sunlight to branches, each with three or four leaves and one capsule, at 35-40 days post-anthesis, inside poly(ethy1ene)bags. After 30 minutes, -80% of the original radioactivity in the bags had been taken up by the branches which were, however, left inside the bags for another 90 minutes. The latter were then removed, and the plants were kept under normal day and night conditions, when photosynthesis could occur normally, until the capsules were harvested. The radioactivity was determined in the fractions 80%soluble in methanol ( O ) ,and (1+3)-@-~-glucan(callose) (O),and (1-*4)-p-D-glucan (cellulose) (a) fractions of the fibers. Numbers in parentheses are the percent radioactivity in the callose and cellulose type of Dglucans, respectively.]

BIOSYNTHESIS OF CELLULOSE

141

nical difficulties with obtaining a clear chase in vivo using intact plants made it hard to decide from these studies whether a quantitative conversion of radioactivity from (1+3)-p-D-ghcan into cellulose had occurred. In the results of Maltby and coworkers,151the chase was effective, as evidenced by a cessation of incorporation of label into cellulose, and in those experiments no turnover was observed. Other cytochemical studies of developing cotton-fibers by Waterk e ~ n indicated '~~ that (1+3)-P-~-glucanis always localized, independent of the age of the fiber, in the innermost wall-layer. The basis of identification of the (1+3)-j%~-glucan was that it showed a specific fluorescence after staining with Aniline Blue, a procedure that is not always specific for (1+3)-P-D-gl~can.'~~ However, Waterkeyn is experienced with the use of this dye, and, assuming that his identification was correct, such a localization for the (1+3)-P-~-glucancould be interpreted to indicate a precursor function. Alternatively, Waterkeyn also offered the suggestion that the glucan could play a role in providing a matrix wherein microfibrils undergo "maturation" and orientation. By chemical analyses of levels of (1+3)-p-~-glucan, Maltby and coworker^'^' found that the maximum level of accumulation occurred during a time when an overlap takes place between the phases of fiber elongation and secondary-wall synthesis, and they similarly suggested that the role of the (1+3)-j3-~-glucancould be in modulating the extensibility of the wall, rather than as a precursor to cellulose. Nevertheless, there are peculiarities in the pattern of labeling of this glucan in vivo that are not readily explained, except by invoking turnover; but, even if such turnover occurs, a specific conversion into cellulose has by no means been proved as yet. A curious analogy in A. rylinurn to the whole (1+3)-P-~-glucanpuzzle in higher plants may also be worth mentioning. In addition to catalyzing the synthesis of (1+3)-/?-D-glucan from UDP-glucose, A. xyZinum membrane-preparations are also active in catalyzing the synthesis from UDP-glucose of water-soluble ( 1 + 2 ) - ~ - ~ - g l u c a n . " ~ * ~ ~ ~ Such a glucan has not been described as a structural component of this organism, although it has been reported to exist in other Grsm-negative bacteria (see references cited in Ref. 112). It has also been observed that some synthesis of this glucan is found in labeling studies in v i ~ dhowever, ~ ~ ; no data as yet exist to support, or refute, an intermediate role for this polymer in cellulose biosynthesis. In summary, much incomplete evidence has been given that offers (156) L. Waterkeyn, Protoplasma, 106 (1981) 49-67. (157) M. M. Smith and M. E. McCully, Protoplasma, 95 (1978) 229-254.

142

DEBORAH P. DELMEH

suggestions for a role for high-molecular-weight precursor(s) to cellulose. Taken in sum, the evidence is not yet fully convincing, nor does it preclude such a possibility. It is, perhaps, useful to consider the implications for biosynthesis that the existence of such a polymer would have. Based on current concepts of mechanisms of polysaccharide synthesis, it is difficult to envisage a mechanism of microfibril synthesis involving such polymers. If, as Colvin' believes, such precursors associate without the aid of enzymes, to afford crystalline cellulose, it is exceedingly difficult to explain three things: (1 ) as cellulose I1 has a structure much more stable than that of cellulose I, it would be expected that spontaneous crystallization of a soluble form would yield cellulose 11, and yet we know that the native cellulose is cellulose I; ( 2 )the d.p.ofthe cellulose would be expected to be low, and we know that it is not; Colvin' proposed the possible existence of chain ligases, but no evidence exists for such enzymes as of this writing; and ( 3 ) whatever polar group exists that must confer solubility to such an intermediate glucan must be removed, a process that would surely require some kind of enzyme. If, on the other hand, transfer of glucan chains from a (protein?) carrier is mediated by an enzyme, it is difficult to envisage why a process of elongation involving carrier-mediated transfer of oligosaccharides should require such long oligosaccharides, instead of D-glucose or cellobiose. Perhaps, a more likely possibility is that such polymers are primers that are subsequently elongated by a different mechanism, to afford the final D-glucan chains in cellulose. With respect to the possibility that (1+3)-P-D-glucan might serve as an intermediate in cellulose synthesis in higher plants, transfer of Dgliicosyl groups to cellulose by trans-D-glucosylation could 6e envisaged; the spontaneous crystallization of the cellulose might serve to drive this reaction in the appropriate direction. Exoglucanases having transglucosylase activity are known to exist, but the new linkages generated are often random. Meier's group'j5 demonstrated the existence in cotton fibers of a wall-bound exoglucanase that has a preference for (1--d)-~-D-giucansas substrate, but they have yet to demonstrate any trans-D-gliicosylase activity for this enzyme. One attractive argument for the hypothesis that this glucan is a precursor to cellulose is that it could nicely explain the rapid production of (l+d)-P-D-glucan that occurs on wounding of plant cells; if the final conversion of this glucan into cellulose is catalyzed by a very labile enzyme-system, it might be expected that, on cellular damage, the precursor would rapidly accumulate. However, much more study is needed before such a possibility can be considered to be more than just speculation.

BIOSYNTHESIS OF CELLULOSE

143

4. Genetic Mutations, and Chemical Inhibitors of

Cellulose Biosynthesis

The elucidation of metabolic pathways has often been aided by the use of genetic mutations, or specific, chemical inhibitors that result in the blockage of a specific, metabolic reaction along the pathway; such inhibition can sometimes lead to the accumulation of prior intermediates, and thus facilitate their isolation and identification. Mutants of A. xylinum that are incapable of, or impaired in their, ability to synthesize cellulose have been isolated. One such mutant was partially characterized by Swissa and coworker^.^ Although this mutant lacked the capacity to synthesize cellulose, it possessed capability for the normal metabolism of hexose phosphates and UDP-glucose; of considerable interest was the observation that, during in vivo labeling with radioactive D-glucose, it showed enhanced accumulation of label in chloroform-, water-, and alkali-soluble material (as compared to the wild type). Such a finding supports a possibility proposed by these workers, namely, that the water- and alkali-soluble fractions may constitute precursors to cellulose. However, a chemical characterization of these fractions in the mutant strain has yet to be reported. They also found that the mutant is still capable of catalyzing the synthesis, from UDP-glucose, of D-glucans in vitro; this is in contrast to results with another mutant studied by Cooper and M a n l e ~ , ' ~ ~ who found their mutant strain incapable of D-glucan synthesis from UDP-glucose. Incorporation, in whole cells, of label from labeled D glucose into the lipid fraction was also lessened in this mutant. Garcia and also mentioned that they observed a lower capacity for glucolipid synthesis from UDP-glucose with another cellulose-less mutant. Unfortunately, the lack of a good genetic system for A. xylinum has made genetic analyses of these mutants impossible to date; in none of the foregoing was it known how many mutations were involved, or where the specific blocks occurred in these metabolic pathways. To the best of the present author's knowledge, there are, unfortunately, in the algae or higher plants, no known mutants available that are specifically blocked in cellulose synthesis. However, several, relatively specific, chemical inhibitors of the process have been characterized. One of these, coumarin, has been reported to inhibit cellulose synthesis in A. x y Z i n ~ m , ' ~as - 'well ~ ~ as in higher plant^?^,'^^ Relatively (158) D. Cooper and R. S. J. Manley, Biochim. Biophys. Acta, 381 (1975) 109-119. (159) S. Satoh, M. Takahama, and K. Matsuda,Plant Cell Physiol., 17 (1976) 1077-1080. (160) J. Burgess and P. J. Linstead, Planta, 133 (1977)267-273.

144

DEBORAH P. DELMER

high concentrations (in the millimolar range) are required in order to obtain a substantial inhibition; this appears to be relatively specific for cellulose synthesis, at least in comparison to its effects on the synthesis of other cell-wall polymers. Coumarin has, however, been reported to have some other side effects in plants, as discussed by Montezinos and Delmer.85Nevertheless, it is of some interest that it was effective in inhibiting the synthesis of proposed intermediates in cellulose synt h e ~ i s , ' ~as~ discussed ,'~~ in Section V,3. Some indication that Mg2+may be an important ion for cellulose synthesis comes from studies by Montezinos and as discussed earlier (in Section IVJ). Also of interest is the observation by Quader and RobinsonBdthat calcium ionophores and the cryptates 211 and 212 were potent inhibitors of cellulose synthesis in Oocystis, although their mode of action is at present not understood. One of the most promising inhibitors studied to date is 2,6-dichlorohenzonitrile (DCB), which has been marketed as a herbicide under the names of Casoron and Dichlobenil. Hogetsu and coworkers'61first provided an indication that the mode of action of DCB as a herbicide could be due to its effect on cellulose biosynthesis in plants. Subsequently, Montezinos and DelmeP showed it to be a specific and reversible inhibitor of cellulose synthesis, effective in low concentrations (1-10 pM)in cotton fibers. Meyer and Herthls2also found DCB to be an effective and reversible inhibitor of cell-wall regeneration in tobacco protoplasts. Aloni and Benziman'O reported that DCB also inhibits cellulose synthesis in A. xylinum. Further studies, with cotton fibers and soybean cells,lm indicated that DCB does not inhibit mglucose uptake, or the synthesis of hexose phosphates or UDP-glucose, nor does it affect ATP levels. However, attempts to demonstrate a DCB-induced accumulation of any intermediates beyond the level of UDP-glucose were not successful. Montezinos and Delmef15pointed out that use of this inhibitor for studies of cellulose synthesis should be confined to short-term experiments, as some indication exists that DCB can be metabolized to a derivative that can affect oxidative phosphorylation.lM The documented, herbicidal activity of DCB offers some promise that inhibition of the process of cellulose synthesis could be further exploited as a safe and effective target of herbicide action. (161) T. Hogetsu, H. Shibaoka, and H. Shimo-Koriyama, Plant Cell Physiol., 15 (1974) 389-393. (162) Y. Meyer and W. He&, Plantn, 142 (1978) 253-262. (163) N. C. Carpita, A. Klein, and D. P. Delmer, unpublished results. (164) D. E. Moreland. G . G . Hussey, and F. S. Fanner, Pestic. Bwchem., Physiol., 4 (1974) 356-364.

BIOSYNTHESIS OF CELLULOSE

145

5. Possible Factors Affecting the Lability of the Polymerizing System

From the preceding discussions, it is evident that, in all systems studied, and, in particular, in higher plants, attempts to synthesize cellulose in vitro have met with only limited success; this therefore leads to the conclusion that, for poorly understood reasons, the cellulose synthetase complex is a highly labile system. As a conclusion to this article, it may prove useful for future research to discuss possible reasons for this apparent lability. a. Effect of Proteases.-One obvious possibility is that the complex is highly susceptible to proteolytic attack. Chao and Ma~lachlan'~~ reported that, present in extracts of pea seedlings was an endogenous factor, suggested to be a protease, that caused partial inactivation of UDP-glucose:(l4)-/3-D-glucan synthetase activity. (As discussed earlier, it is not certain whether this enzyme functions in synthesis of cellulose or of xyloglucan.) Nevertheless, attempts by these workers to prevent the inactivation by the addition of protease inhibitors or high concentrations of nonspecific protein were unsuccessful. Through conversations with colleagues in the field, as well as personal experience, it is clear that numerous attempts to inhibit protease activity have not resulted in a substantial enhancement of UDP-glucose:(1+4)-p-~glucansynthetase activity. Likewise, the present author knows of no successful attempts to stimulate activity by limited protease treatment, a procedure used with great success for chitin ~ynthetase'~*'~' (EC 2.4.1.16). b. Effect of Poly(ethy1ene Glycol).-It has observed that inclusion of 0.06 molal poly(ethy1ene glycol)-4O00 (PEG-4000) in the isolation medium results in a considerable enhancement of UDP-g1ucose:glucan synthetase activities in membrane preparations derived from cotton fiber~.l@J~~ Polymerization of both &( 1+3)- and p-( 14)-glucosyl residues is enhanced; whereas, in previous work,3l it was possible to detect synthesis only of (1+3)-p-~-glucan,from UDP-glucose in the (165) H.-Y. Chao and G. A. Maclachlan, Plant Physwl., 61 (1978) 943-948. (166) E. Cabib, R. Ulane, and B. Bowers, in B. L. Horecker and E. R. Stadtman (Eds.), Current Topics in Cellular Regulation, Vol. 8, Academic Press, New York, 1974, pp. 1-32. (167) J. Ruiz-Herrera, E. Lopez-Romero, and S. Bartnicki-Garcia,J . Biol. Chem., 252 (1977) 3338-3343. (168) D. P. Delmer, M. Benziman, A. Klein, A. Bacic, B. Mitchell, H. Weinhouse, Y. Aloni, and T. Callaghan,]. Appl. Polym. Sci., in press. (168)A. Bacic and D. P. Delmer, Planta, 152 (1981)346-351.

146

DEBORAH P. DELMER

presence of PEG4000, it is now routinely observed that -25% of the glucan products contain p-( 14)-glucosyl residues.169Poly(ethy1ene glycols) of lower molecular weight were less effective. A similar enhancement of activity (- 10-fold) by PEG4000 has also been observed for the A. xylinum UDP-glucose:( 1+4)-p-~-glucansynthetase."jRMaclachlan and coworkers170also observed some enhancement of UDPglucose:( 1+4)-P-~-glucan synthetase from pea tissue on using PEG400. Inclusion of PEG in isolation buffers may, therefore, prove to be of considerable help in stabilizing such enzymes. Poly(ethy1ene glycol)~ of high molecular weight are known to promote protein-protein and this could be the mechanism whereby these substances stabilize the activity of a multi-subunit enzyme-complex. It should also be noted, however, that inclusion of PEG4000 in the isolation buffer leads to the production of abnormally large, membrane vesicles that sediment at low centrifugal forces, presumably due to the known ability of PEG to promote membrane fusions. At present, it is not known whether these vesicles contain components only from the plasma membrane, or represent mixed-membrane fusions. c. Attempts to Assay Solubilized Glucan Synthetases-Some attempts to solubilize and purify a UDP-glucose:( 1+4)-p-D-glucan synthetase activity have been made. Tsai and H a ~ s i dwere ' ~ ~ able to solubilize p-( b 3 ) - and p-( l-A)-glucan synthetase activities from membranes of oat seedlings by use of high concentrations of digitonin; they were also able to resolve these activities by chromatography in a column ofhydroxylapatite; however, the solubilized enzymes were quite unstable, and further purification was not attempted. Larsen and BnLmmond'i4 also succeeded in solubilizing, with digitonin, these activities from membranes of Lupinus albus; however, no puriachieved good solubilization of fication was attempted. Klein16H-1i5 these activities from membranes derived from cultured soybean cells; her soliibil ization procedure involved treatment of the membranes for 15 minutes at 0" with 30 mM cholate at pH 7.8. The solubilized enzymes were quite labile, but could be both stimulated and stabilized by high concentrations of glycerol. Advances in solubilization and re(170) G. Maclachlan, M. Durr, and Y. Raymond,Methodol. Sum. (B)Biochem., 9 (1979) 147-153. (171) L. A. Halper and P. A. Srere, Arch. Biochem. Biophys., 184 (1977) 529-534. (172) J. C. Lee and L. L. Y. Lee,J. Biol. Cliem., 256 (1981)625-631. (173) C. M. Tsai and W. Z . Hassid, Plont Physiol., 47 (1971) 740-744. (174) G. L. Larsen and D. 0. Brurnrnond, Phytochemistry, 13 (1974)361-365. (175) .4. S. Klein, Ph.D. Thesis, Michigan State University, 1981.

BIOSYNTHESIS OF CELLULOSE

147

constitution of membrane-bound proteins into artificial lipid vesicles suggest that it would be profitable to pursue these preliminary studies farther. However, to date, no dramatic increases in activity for UDPglucose:( 1+4)-P-D-glucan synthetase have been reported as a result of working with a solubilized form of the enzyme. d. Requirement for an Intact Cell for Activity; Possible Modulation of Activity by a Transmembrane, Electrical Potential.-Assay of cel-

lulose synthetase activity in intact cells has been attempted in a number of laboratories. In higher plants, incubation of pea-epicotyl slices in radioactive UDP-glucose results in production of P-glucan, but the * ~ ~ , ~ ~ ~ and Ray115conproduct is mainly ( 1 + 3 ) - P - D - g l u ~ a n . ~Anderson cluded that this activity occurred primarily at the cut edges of the tissue. Similarly, Delmer and coworkers1lsobserved that intact cottonfibers do not utilize UDP-glucose, but substantial activity for synthesis of (1-*3)-P-D-glUCan is obtained when the cells are damaged; Brett176found a similar phenomenon with suspension-cultured, soybean cells. Klein and Delmel.26*175 observed that preparations of “intact,” soybean protoplasts could utilize UDP-glucose for synthesis of (1+3)-P-D-glucan, but activity was enhanced at least 10-fold when the protoplasts were lysed; therefore, it was difficult to exclude the possibility that the “intact” protoplast-preparation contained a low percentage of damaged protoplasts. The main conclusions from all of these experiments are that (a) intact plant-cells utilize UDP-glucose poorly, or not at all; and (b) when damaged (or even just rendered permeable by treatment with a detergent or dimethyl sulfoxide, or by cold shock”’), they can utilize the substrate for synthesis of (1+3)-P-D-glucan, but not for synthesis of cellulose. This, in turn, leads to the conclusion that the cellulose synthetase complex can only accept UDPglucose from the inner face of the plasma membrane and, unfortunately, loss of cellular integrity results in inactivation of the complex. Carpita and Delmer177*178 proposed that one feature of an intact cell, that is, a transmembrane, electrical potential (A$) may be an important factor for maintaining an active, cellulose-synthetase complex. This hypothesis evolved from observations of another effect of PEG-4000 on cellulose synthesis in cotton fibers; it was found that cutting of intact fibers (just once) with scissors resulted in an essentially total cessation of the synthesis of radioactive cellulose from radioactive D-ghCOSe supplied. [The synthesis of (1+3)-p-D-glucan, was not, however, substantially lessened, suggesting that energy-generating systems neces(176) C. T. Brett, Plant Physiol., 62 (1978) 377-382. (177) N. C. Carpita and D. P. Delmer, Plant Physiol., 66 (1980)911-916, (178) N. C. Carpita, in Ref. 10, pp. 225-242.

148

DEBORAH P. DELMER

n-n- L n :ontrol

K - MES

4L

K-l

SIVAL

(+)

BTP/N03 (-)

Frc. 5.-Stimulation of UDP-g1ucose:glucan Synthetase Activities by Conditions that Lead to Induction of a Transmembrane, Electrical P0tentia1.l~~ {The experiment was performed by using membrane vesicles prepared from developing cotton-fibers;

into total P-D-glUCanS was meaincorporation of radioactivity from UDP-~-[~~C]glucose sured. Anion and cation concentrations were 50 mM; valinomycin (VAL) was present at 5 p M ;and UDP-glucose at 0.1 mM; 1 pCi per pmol.}

sary for synthesizing activated Dglucose substrates were not seriously impaired by this procedure.] However, if the fibers were cut in the presence of 0.06 molal PEG-4000, the rate of cellulose synthesis observed was 50% of that of uncut fibers. A variety of observations led to the conclusion that this protective effect of PEG is mainly due to a promotion of resealing of the cut fiber-membranes, which thereby results in restoration of an “intact” cell. Thus, it was reasoned that some feature of an intact cell was essential for maintaining active synthesis of cellulose. It was possible to rule out turgor pressure, and to propose, instead, that a transmembrane, electrical potential could be the critical factor required. Results of later studies by Delmer and coworker^^^^*^^^ provided some support for this hypothesis. Thus, it was shown that re-establishment of a transmembrane, electrical potential (positive-inside) across vesicles isolated from cotton fibers resulted in a 4-12-fold stimulation of p-D-glucan synthesis from UDP-glucose (see Fig. 5). Such a potential was established by the addition of K+ in the presence of an imper-

-

(179) D. P. Delmer, N. C. Carpita, A. Bacic, and D. Montezinos, Proc. Ekman-Days Int. Symp. Wood Pulp. Chern., 3 (1981) 25-27.

BIOSYNTHESIS OF CELLULOSE

149

meant anion, such as 2-(4-morpholino)ethanesulfonicacid (MES), to membrane vesicles incubated in the presence of valinomycin (VAL). VAL is a K+-specific ionophore that allows the free movement of K+ down its concentration gradient, thereby establishing a net, positive-inside potential. Conditions that should lead to a negative-inside potential, such as the addition of an impermeant cation, for example, 1,3-bis[tris(hydroxymethyl)methylamino]propane (“bistrispropane”; BTP) together with a permeant anion such as NO3-, resulted in no substantial stimulation. Other experiments provided further evidence that the effect was truly due to a creation of A$, and not just to stimulation by K+; it was also shown that creation of a ApH across the vesicles did not lead to stimulation; only creation of A$ (positive-inside) was successful. It is of interest that only a positive-inside potential was effective. As UDP-glucose can presumably only be accepted as substrate from the inner face of the plasma membrane, it is presumed that only inverted vesicles are active; therefore, a positive-inside potential in an inverted vesicle mimics the in vivo situation, which is negative-inside. Analyses of the linkages found in the products of the stimulated reaction revealed that polymerization of both p-( 1+3)- and p-( 14)-glucosyl residues was enhanced. Once again, it is necessary to point out that no proof exists at present that this UDP-glucose:(14)-p-&glucan synthetase activity is related to a true, cellulose-synthesizing reaction. Results of Delmer and coworkers,’80with A. xylinum also indicated that the existence of a A$ across the cell membrane may be a crucial factor for maintaining active synthesis of cellulose. In these studies, it was shown that dissipation of A$, by addition of K+ and VAL to EDTA-treated, A. xylinum cells in the presence of the impermeant anion S042-, resulted in an essentially complete inhibition of cellulose synthesis from supplied D-glucose. When the experiments were performed under conditions where energy metabolism could still be driven18’ by a transmembrane ApH (that is, at pH 4.5;ApH -1.3 or 76 mV), the effect of dissipating A$ was specific for cellulose synthesis; it was also reversible, because, when cells were transferred from a high-K+ medium to a high-Na+ medium, cellulose synthesis resumed. Several possible mechanisms can be envisaged to explain how A$ may modulate the activity of the cellulose-synthetase complex. The effects of membrane fluidity on membrane-bound enzymes is well (180) D. P. Delmer, M. Benziman, and E. Padav, Proc. Natl. Acad. Sci USA, 79 (1982) 5282-5286. (181) E. D. Padan, D. Zilberstein, and H. Rottenberg,Eur. J . Biochern., 63 (1976) 533541.

1*50

DEBORAH P. DELMER

documented.’”2 Lelkes’s:’ has shown that changes in AJ, markedly influence the fluidity (and, almost certainly, also, the orientation of lipids) in phospholipid vesicles. Thus, it is quite conceivable that changes in the lipid environment surrounding a highly organized, enzyme complex could result in conformational changes in the complex. Another possibility is that changes in A+ could influence the movement of substrates (UDP-glucose, lipid intermediates, or proteinlinked carriers) within the membrane. In any case, the loss of A+ upon cellular disruption could well be one factor responsible for the loss of enzyme activity in citru. VI. CONCLUSIONS Many difficulties have been encountered in the study of the biosynthesis of cellulose, chief among them being the apparent lability of the cellulose-synthetase system. Based on evidence accumulated to date, a current model of cellulose synthesis184can be envisaged as indicated in Fig. 6. Polymerization of the mglucan chains occurs by way of a multi-subunit, enzyme complex embedded in the plasma membrane; an almost simultaneous association, by means of hydrogen bonds, of the newly formed chains results in formation of partially crystalline microfibrils. This mechanism of polymerization and crystallization results in the creation of microfibrils whose chains are oriented parallel (cellulose I). In A. xylinurn, the complex is apparently immobile, but, in cells in which cellulose is deposited as a cell-wall constituent, it seems probable that the force generated by polymerization of the relatively rigid microfibrils propels the complex through the fluid-mosaic membrane. The direction of motion may be guided through the influence of microtubules. Much controversy has surrounded the question as to the nature of the active form(s) of D-glucose that serve(s) as precursor to cellulose. Current evidence strongly favors a role for UDP-glucose; much suggestive, but by no means conclusive, evidence indicates that lipid- or protein-linked intermediates, or both, may also be involved. Much of the difficulty in studying cellulose biosynthesis may be at(182) H. K. Kimeiberg, Cell Surf. Rev., 3 (1977)205-293. (183)P. I. Lelkes, Biochem. Biophys. Res. Commun., 90 (1979)656-662. (184)D.P. Delnier, in C. C. Black and A. Mitsui (Eds.), CRC Handbook Series ofBiosolar Resources, Vol. I : Basic Principles, CRC Press, Boca Raton, Florida, 1982, pp. 351-355.

Cell wall

1

BIOSYNTHESIS OF CELLULOSE

u\

Plasma membrane

151

Cytoplasm

Mobile cellulose svnthetase

r

D- gtucose 6 - P

\I

0-

-

glucose I P

Individual D-glucan chain

.UDP

w

fructose Growing microfibril

microtubules?) involved in the orienting movement

sucrose

FIG.6.-Hypothetical Model for the Biosynthesis of [Numbers refer to reactions catalyzed by the following enzymes: 1, invertase (EC 3.2.1.26); 2, sucrose synthetase; 3, hexokinase (EC 2.7.1.1); 4, phosphoglucomutase (EC 2.7.5.1); 5, UDPglucose pyrophosphorylase; and 6, 7, and 8, hypothetical reactions in the pathway to cellulose.]

tributed to the apparent lability of the synthesizing complex. Such compounds as glycerol or PEG4000 have been found to offer some protection of UDP-glucose:( ld)-p-D-ghcan synthetase activity. A hypothesis that the activity may be modulated by the transmembrane, electrical potential may offer another clue to the lability of the complex in vitro. Nevertheless, it is clear that many questions concerning the nature of the complex, and of the process in general, remain unanswered. The writing of such an article as this is a tedious process; but, if even just one imaginative young scientist is stimulated to join the quest as a result of reading it, the effort will have been well worth while. The study of cellulose biosynthesis requires both a combination of careful scientific analyses and imagination, and the field can only profit from the entry of more scientists possessing these qualities. An invitation is extended to you to join in.

152

DEBORAH P. DELMER

VII. ADDENDUM Since this article was sent to press, several noteworthy findings have been reported in the literature. The first concerns the structural identification of the radioactive compounds which appeared to serve as precursors to cellulose inA. xylinum (see Fig. 3). In a note added in proof to Ref. 168, Delmer and coworkers have concluded that the radioactive compounds present in the water- and alkali-soluble fractions analyzed by Swissa and coworkerss and Aloni and Benziman’O consist of a mixture of sugar phosphates (Dfructose 1,6-bisphosphate7 Dglucose 6-phosphate7and Dfructose &phosphate) and fine fibrils of cellulose. During a chase with unlabeled Dglucose, labeled carbon from the sugar phosphates is rapidly converted into cellulose, concomitant with a much slower, apparent “chase” of the fine fibrils of cellulose into the mat of larger aggregates of cellulose produced upon prolonged incubation in high concentrations of Dglucose. Thus, Delmer and coworkers168concluded that, as a result of a quite extensive analysis of the chloroform-methanol-soluble, water-, and alkali-soluble fractions, no positive evidence exists for intermediates beyond the level of UDP-glucose in A. xylinum. The second finding concerns reports by Aloni and coworkers185and Benziman and coworkersla6 of success in achieving high rates of in vitro synthesis of (1+4)-&~glucanfrom UDP-glucose by using membrane preparations derived from A. xylinum. The key to this success lay in the discovery that the A. xylinum enzyme-system can be activated by GTP. Activation by GTP requires the presence of an additional, protein factor; this factor tends to dissociate from the enzyme, but enzyme-factor association can be promoted by PEG-4000 or by Ca*+.Under optimal conditions, that is, in the presence of GTP, factor, and PEG4000 (or Caz+),initial rates of synthesis of (l-;.4)-P-~-glucan that are 200 times higher than any previously reported can be achieved; such rates exceed 40% of the in viuo rate of synthesis of cellulose in A. xylinum. The enzyme system has also been successfully solubilized by using digitonin, and the solubilized enzyme possesses high activity and displays all of the regulatory properties observed for the membrane-bound enzyrne.l8’ These findings offer new hope that future, in uitro studies can lead to a detailed understanding of the (185) Y. Aloni, D. P. Delmer, and M. Benziman, Proc. Natl. Acad. Sci. USA, (1982) in press. (186) M. Benziman, Y. Aloni, and D. P. Delmer, J . A p p l . Polym. Sci., (1982)in press. (187) Y. Aloni, M. Benziman, and D. P. Delmer,J. BWL. Chem., (1983) in press.

BIOSYNTHESIS OF CELLULOSE

153

mechanism and regulation of the synthesis of cellulose in A. xylinum. It will certainly also be of interest to know if the activation by poly(ethy1ene glyco1)s of plant D-ghcan synthetases (see Section V, 5,b) relates to a similar, regulatory mechanism for the synthesis of cellulose in higher plants.