On the alignment of cellulose microfibrils by cortical microtubules: A review and a model

Summary The hypothesis that microtubules align microfibrils, termed the alignment hypothesis, states that there is a causal link between the orientation of cortical microtubules and the orientation of nascent microfibrils. I have assessed the generality of this hypothesis by reviewing what is known about the relation between microtubules and microfibrils in a wide group of examples: in algae of the family Characeae,Closterium acerosum, Oocystis solitaria, and certain genera of green coenocytes and in land plant tip-growing cells, xylem, diffusely growing cells, and protoplasts. The salient features about microfibril alignment to emerge are as follows. Cellulose microfibrils can be aligned by cortical microtubules, thus supporting the alignment hypothesis. Alignment of microfibrils can occur independently of microtubules, showing that an alternative to the alignment hypothesis must exist. Microfibril organization is often random, suggesting that self-assembly is insufficient. Microfibril organization differs on different faces of the same cell, suggesting that microfibrils are aligned locally, not with respect to the entire cell. Nascent microfibrils appear to associate tightly with the plasma membrane. To account for these observations, I present a model that posits alignment to be mediated through binding the nascent microfibril. The model, termed templated incorporation, postulates that the nascent microfibril is incorporated into the cell wall by binding to a scaffold that is oriented; further, the scaffold is built and oriented around either already incorporated microfibrils or plasma membrane proteins, or both. The role of cortical microtubules is to bind and orient components of the scaffold at the plasma membrane. In this way, spatial information to align the microfibrils may come from either the cell wall or the cell interior, and microfibril alignment with and without microtubules are subsets of a single mechanism.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation.

The arrangement of cortical microtubules (MTs) in differentiating tracheids of Abies sachalinensis Masters was examined by confocal laser scanning microscopy after immunofluorescent staining. The arrays of MTs in the tracheids during formation of the primary wall were not well ordered and the predominant orientation changed from longitudinal to transverse. During formation of the secondary wall, the arrays of MTs were well ordered and their orientation changed progressively from a flat S-helix to a steep Z-helix and then to a flat S-helix as the differentiation of tracheids proceeded. The orientation of cellulose microfibrils (MFs) on the innermost surface of cell walls changed in a similar manner to that of the MTs. These results provide strong evidence for the co-alignment of MTs and MFs during the formation of the semi-helicoidal texture of the cell wall in conifer tracheids.Abbreviations MT cortical microtubule - MF cellulose microfibril - S1, S2 and S3 the outer, middle and inner layers of the secondary wall The authors thank Mr. T. Itoh of the Electron Microscope Laboratory, Faculty of Agriculture, Hokkaido University, for his technical assistance. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture, Japan (no. 06404013).     

Orientation of cellulose microfibrils in cortical cells of tobacco explants

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations — all given with respect to the longitudinal axis of the cell —, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.Abbreviations CMF cellulose microfibril - MT microtubule We thank Professor M.M.A. Sassen and Dr. G.W.M. Barendse (Department of Experimental Botany, University of Nijmegen, Nijmegen, The Netherlands) for helpful discussions and Mrs. A. Kemp for her assistance in the ethylene experiments.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

Arrangement of cortical microtubules at the surface of the shoot apex inVinca major L.: Observations by immunofluorescence microscopy

Arrangements of cortical microtubules (MTs) and of cellulose microfibrils at the surface of the vegetative shoot apex ofVinca major L. were examined by immunofluorescence microscopy and polarizing microscopy, respectively. Cortical MTs adjacent to the outermost walls of the apex were arranged more or less randomly in individual cells: especially in cells in the central region of the apex the arrangement was almost completely random. However, in the peripheral region MTs tended to show parallel alignment in individual cells, and an overall pattern that was roughly concentric around the apical dome was discerned. Observations of birefringence of cell walls indicated that cellulose microfibrils in the peripheral region of the apex were also arranged in a pattern which was roughly concentric around the apical dome. These patterns of arrangements of MTs and microfibrils are understood to be perpendicular to the radial cell files observed in the peripheral region of the apex, and can be related to the radial expansion of the surface of the apex.     

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements

Summary. The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.Correspondence and reprints: Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881, U.S.A.Present address: Biology Editors Co., Peacedale, Rhode Island, U.S.A.Present address: Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, U.S.A.Present address: Department of Crop Science and Department of Botany, North Carolina State University, Raleigh, North Carolina, U.S.A.     

Helicoidal orientation of cellulose microfibrils in Nitella opaca internode cells: ultrastructure and computed theoretical effects of strain reorientation during wall growth

The ultrastructure of the mature internode cell wall of Nitella opaca is described. It is interpreted in terms of a helicoidal array of cellulose microfibrils set in a matrix. A helicoid is a multiple plywood made up of layers of parallel microfibrils. There is a progressive change in direction from ply to ply, giving rise to characteristic arced patterns in oblique sections. A critical tilting test, using an electron microscope fitted with a goniometric stage, showed the expected reversal of direction of the arced pattern. Nitella cell wall is thus more regularly structured than previous studies have shown. From a survey of the cell-wall literature, we show that such arced patterns are common. This indicates that the helicoidal structure may be more widespread than is generally realised, although numerous other cell walls show no signs of it. Nevertheless, there are examples in most major plant taxa, and in several types of cells, including wood tracheids. Most of the examples, however, need confirmation by tilting evidence. There are possible implications for wall morphogenesis. Helicoidal cell walls might arise by selfassembly via a liquid crystalline phase, since it is known that the cholesteric state is itself helicoidal. A computer graphics programme has been developed to plot the expected effects of growth strain on the patterns in oblique sections of helicoids with various original angles between consecutive layers. Herringbone patterns typical of crossed polylamellate texture can be generated in this way, indicating a possible mode of their formation.     

Regulation of the orientation of cortical microtubules inSpirogyra cells

The orientation of cortical microtubules (MTs) was synchronously regulated inSpirogyra cells. While the reorganized MTs in distilled water for 1.5 hr, after 1 hr treatment with amiprophos-methyl (APM) and complete depolymerization of the MTs, were all transverse, those reorganized in 0.30 M mannitol were all oblique or longitudinal. After the MTs had reorganized in 0.30 M mannitol, these cells were then incubated in distilled water for 6 hr, and the orientation of the MTs, in the cells in which MTs could be observed, all became transverse.     

Deposition and reorientation of cellulose microfibrils in elongating cells of Petunia stylar tissue

According to Roelofsen and Houwink's (1953, Acta Bot. Neerl. 2, 218–225) multinet growth hypothesis, microfibrils originally deposited transversely in the cell wall become gradually reoriented towards more axial orientations during cell elongation. To establish the extent of reorientation, microfibrils were studied during their deposition and elongation, using stylar parenchyma and transmitting tissue cells of Petunia hybrida L. At the inner surface of very young cells, microfibrils were deposited in alternating Z- and S-helical orientations. The following sequence in deposition, from the exterior to the interior side of the wall, could be inferred: Axial: 150°–180° (Z-helical), 0°–30° (S-helical); oblique: 110°–150° (Z-helical), 30°–70° (S-helical); transverse: 90°–110° (Z-helical), 70°–90° (S-helical). With the increasing pitch, the density of the deposited microfibrils increased as well, giving rise to an alternating helical texture. During elongation, only transversely S- and Z-helically oriented microfibrils were deposited and all microfibrils underwent a certain reorientation as described in the multinet growth hypothesis. The texture resembled that of young cells and the wall maintained its thickness. The extent of passive reorientation was in agreement with the theoretical calculations made by Preston.Dedicated to Professor Dr. A.B. Wardrop, Melbourne, on the occasion of his 70th birthday     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Root contraction in hyacinth IV. Orientation of cellulose microfibrils in radial longitudinal and transverse cell walls

Summary Cellulose microfibrils (MFs) were visualized on the inner surface of root cortex cell walls ofHyacinthus orientalis L. using a replica technique. Microfibril orientation was determined in radial longitudinal and transverse cell walls of the root tip, uncontracted, contracting, and fully contracted regions of the root. In longitudinal walls, the innermost MFs were ordered and parallel to one another and were oriented transversely, axially or obliquely, depending upon the developmental stage of the region. In transverse walls MFs in a single layer formed crisscross or ordered parallel arrays, depending upon the region. Parallel arrays were oriented either parallel, perpendicular, or oblique to the radius of the root. Inner walls of certain cells in the contracting region had MFs which appeared interrupted over their lengths. In general, these findings parallel earlier immunofluorescence and electron microscopic observations of changing cortical microtubule (MT) orientation accompanying root contraction. The major exception to MT-MF congruence occurred in cells of the actively contracting region. In middle and outer cell layers, MFs appeared short and partially obscured, while MTs in these cells occurred in conspicuous laterally aggregated strands parallel to one another over the length of the cells or were absent. This alteration in MF-MT parallelism may be related to the reorientation in cell growth occurring in the contractile zone or to the collapse of specific cells during the process of root contraction.Abbreviations MF microfibril - MT microtubule     

Spatial relationship between microtubules and plasma-membrane rosettes during the deposition of primary wall microfibrils in Closterium sp.

The mechanism by which cortical microtubules (MTs) control the orientation of cellulose microfibril deposition in elongating plant cells was investigated in cells of the green alga, Closterium sp., preserved by ultrarapid freezing. Cellulose microfibrils deposited during formation of the primary cell wall are oriented circumferentially, parallel to cortical MTs underlying the plasma membrane. Some of the microfibrils curve away from the prevailing circumferential orientation but then return to it. Freeze-fracture electron microscopy shows short rows of particle rosettes on the P-face of the plasma membrane, also oriented perpendicular to the long axis of the cell. Previous studies of algae and higher plants have provided evidence that such rosettes are involved in the deposition of cellulose microfibrils. The position of the rosettes relative to the underlying MTs was visualized by deep etching, which caused much of the plasma membrane to collapse. Membrane supported by the MTs and small areas around the rosettes resisted collapse. The rosettes were found between, or adjacent to, MTs, not directly on top of them. Rows of rosettes were often at a slight angle to the MTs. Some evidence of a periodic structure connecting the MTs to the plasma membrane was apparent in freeze-etch micrographs. We propose that rosettes are not actively or directly guided by MTs, but instead move within membrane channels delimited by cortical MTs attached to the plasma membrane, propelled by forces derived from the polymerization and crystallization of cellulose microfibrils. More widely spaced MTs presumably allow greater lateral freedom of movement of the rosette complexes and result in a more meandering pattern of deposition of the cellulose fibrils in the cell wall.Abbreviations E-face exoplasmic fracture face - MT microtubule - P-face protoplasmic fracture-face     

Immunofluorescence microscopy of microtubule arrangement in Closterium acerosum (Schrank) Ehrenberg

The microtubule (MT) arrangement in Closterium acerosum cells was observed by indirect immunofluorescence microscopy both during and following cell division, and during cell expansion without cell division. (During the division period, some cells of this alga divide whereas other cells expand in their middle region without division.) Before septum formation, all cells had a ring-like MT bundle (MT ring) in their middle. Both septum formation and expansion without cell division occurred at the position of this ring. During the periods of division, short, hair-like MTs appeared around the nucleus in some of the cells, in addition to the MT ring. In dividing cells, spindle MTs appeared as the chromosomes were condensed. During the early stages of expansion of the semicells, after cell division, the spindle MTs assumed a radial arrangement, moved, and settled in a position between the daughter chloroplasts. These MTs disappeared about 1.5 h after septum formation. As the new semicells were growing, wall MTs appeared, arranged transversely along the expanding wall. These transverse MTs disappeared gradually 4–5 h after septum formation, and only an MT ring remained near the boundary between the new and old semicells. The MT ring was present until the next cell division or expansion without cell division. During the latter course of development, transverse wall MTs were present only at the band-like expanding region. At the earlier stage of expansion without cell division, the short, hair-like MTs remained around the nucleus, but as time passed, both the hair-like MTs and, somewhat later, the transverse ones disappeared and only the MT rings remained. The remaining MT ring was not always positioned at the boundary between the expanding and the old cell region. The temporal relationships between the changes in MT arrangement, and the orientation and localization of cellulose-microfibril deposition are discussed.Abbreviations DAPI 46-diamino-2-phenylindole - EGTA ethyleneglycol-bis-(-aminoethylether)-N, N, N, N-tetraacetic acid - MT mierotubule - PMSF phenylmethylsulfonyl fruoride     

Regional Differentiation in the Dynamics of the Pollen Seasons of Alnus, Corylus and Fraxinus in Poland (Preliminary Results)

In 1995–1996 a study of pollen concentrations of Corylus, Alnus and Fraxinus was performed at seven sites in regions of different climatic and natural conditions. The aim of the study was to determine whether regional differences in the course and duration of pollen seasons occur in Poland. The study was performed using a volumetric method. Several phases during the pollen season were defined for each taxon (1, 2.5, 5, 25, 50, 75, 95, 97.5, 99% of annual total) and duration of the pollen seasons was calculated using 98 and 90% methods. Dynamics and duration of the pollen seasons and a start of particular phases of the season were compared among sites. On the basis of the preliminary analysis it could be supposed that regional differences were most evident in the case of Corylus. The pollen season of this taxon started the earliest in Pozna where thermal conditions were most favourable and the latest in Gdask, a place at the furthest to the north ( ², 0.05). In montane regions (Zakopane, Rabka) last phases of the season were significant extended ( ², 0.05). Probably it results from secondary pollen deposition and a long-distance transport by montane wind. In case of Fraxinus the significant regional differences in the start of the pollen season were not found. The study supported that weather conditions have the substantial influence on the start of consecutive phases of the pollen season.     

Arrangement of cortical microtubules in the shoot apex of Vinca major L.

The arrangements of cortical microtubules (MTs) and of cellulose microfibrils in the median longitudinal cryosections of the vegetative shoot apex of Vinca major L., were examined by immunofluorescence microscopy and polarizing microscopy, respectively. The arrangement of MTs was different in the various regions of the apex: the MTs tended to be arranged anticlinally in tunica cells, randomly in corpus cells, and transversely in cells of the rib meristem. However, in the inner layers of the tunica in the flank region of the apex, cells with periclinal, oblique or random arrangements of MTs were also observed. In leaf primordia, MTs were arranged anticlinally in cells of the superficial layers and almost randomly in the inner cells. Polarizing microscopy of cell walls showed that the arrangement of cellulose microfibrils was anticlinal in tunica cells, random in corpus cells, and transverse in cells of the rib meristem; thus, the patterns of arrangement of microfibrils were the same as those of MTs in the respective regions. These results indicate that the different patterns of arrangement of MTs and microfibrils result in specific patterns of expansion in the three regions. These differences may be necessary to maintain the organization of the tissues in the shoot apex.Abbreviations MT(s) microtubule(s) - lp length of the youngest leaf primordium     

Arrangement of cortical microtubules during formation of bordered pit in the tracheids ofTaxus

Summary The arrangement of cortical microtubules during the development of the secondary wall and bordered pits in the tracheids ofTaxus was examined by immunofluorescence and electron microscopy. The cambial region of radial longitudinal sections of developing young shoots (2–3 years old) contains cells at various stages of differentiation from cambial cells to tracheids. At the early stage of formation of bordered pits, circular bands of microtubules were seen to be associated with the inner edge of the border of the developing pit. In other regions than the pit secondary wall of uniform thickness was laid down, and obliquely oriented cortical microtubules ran parallel to one another. These cortical microtubules also covered the surface of the border of the developing pit on the side facing the center of the cell. As the border of the pit developed, a circular band of MTs remained associated with the inner edge of border, suggesting that the MTs were involved in the formation of the rim of the bordered pit, extending the initial border thickening, which consisted of concentrically oriented cellulose microfibrils. After completion of the formation of the bordered pit, helical thickenings became apparent. The obliquely oriented microtubules were organized in bands parallel to one another, being superimposed on the helical thickenings. The involvement of MTs in the formation of bordered pits and helical thickening is discussed.     

Spatial genetic structure within two populations of a self-incompatible perennial,Chionographis japonica var.japonica (Liliaceae)

Intrapopulational spatial genetic structure was examined in two populations ofChionographis japonica var.japonica, a self-incompatible perennial, by spatial autocorrelation analysis of enzyme polymorphism. Although most spatial autocorrelation indices (Moran'sI) in the shortes distance class were significantly positive, most in the other distance classes did not significantly deviate from the values expected from random distributions of genotypes in both populations. This contrasts with a spatial genetic pattern previously reported for a population of the predominantly selfing congener,C. japonica var.kurohimensis, indicating that pollen-mediated gene flow highly impedes genetic substructuring within populations of outcrossingC. japonica var.japonica. Genetic similarity in very proximate distance found in outcrossingC. japonica var.japonica is probably due to restricted dispersal of seeds.     

Assembly of cortical microtubules during cold treatment of the coenocytic green alga,Chaetomorpha moniligera

The effects of cold treatment on the cortical microtubules (MTs) of Chaetomorpha moniligera Kjellman were investigated by immunofluorescence microscopy. Cortical MTs in Chaetomorpha thallus are arranged longitudinally. In this study, 70–75% of MTs disassembled within 4 h on ice while the others remained stable under these conditions. Reticulate background immunofluorescence, assumed to indicate the presence of a tubulin monomer, was distributed about the stable MTs. Immunofluorescence was prominent in only 50% of the cells. Tubulin polymerization was noted where the background and MT immunofluorescence was strong. New MTs grew transversely as single strings or clusters from the sides of MTs after cold treatment for 4 h and elongated with time to take on a reticulate form at 24 h. The significance of this tubulin polymerization under cold treatment is discussed.Abbreviations MT microtubule - MTOC microtubule-organizing center     

Changes in the non-structural carbohydrate content of cotton (Gossypium spp.) fibres at different stages of development

The neutral sugars (glucose, fructose, and sucrose) and the sugar phosphates (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) soluble in hot aqueous 80% methanol from the fibres of cotton — Gossypium arboreum L., G. barbadense L., and G. hirsutum L. — were determined at various stages of fibre development. In addition, the (13)--D-glucan content was measured and in the case of G. arboreum the rate of (13)--D-glucan and cellulose synthesis was determined with [¹⁴C]sucrose as the precursor. For each of the species a similar chronology was obtained for the changes in content of the various non-structural carbohydrates. At the early stages of secondary wall formation, glucose and fructose exhibited a maximum which was closely followed by a maximum in the (13)--D-glucan content and in the sugar phosphates. On the other hand, the sucrose content increased regularly until fibre maturity. The rates of synthesis of (13)--D-glucan and of cellulose were highest following the maximum in the (13)--D-glucan content, when the latter was being depleted.Abbreviations DMSO dimethyl-sulphoxide - DPA days post anthesis - UDP-glucose uridinediphosphoglucose