Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine.     

β-Glucan hydrolases from Aspergillus niger. Isolation of a β-(1→4)-glucan hydrolase and some properties of the β-(1→3)-glucan-hydrolase components

1. The components of an enzyme preparation from Aspergillus niger, which hydrolysed substrates containing beta-(1-->3)- and beta-(1-->4)-glucosidic linkages, were separated by calcium phosphate and Dowex 1 column chromatography. 2. The hydrolytic activity of each fraction from both types of column towards laminaribiose, laminarin, carboxymethylpachyman, pachydextrins, salicin, cellobiose, cellopentaose and swollen cellulose was tested. 3. The activity towards the beta-(1-->3)-glucosidic substrates was found in three well-separated groups of fractions. The differences in action pattern of these groups is discussed. 4. Preparative-scale chromatography that enabled the separation of a beta-(1-->4)-glucan-glucanohydrolase component substantially free of activity towards beta-(1-->3)-glucosidic substrates is described. Residual beta-(1-->3)-glucan-hydrolase activity was removed by adsorption on to insoluble laminarin at pH3.5.     

Effect of molecular size and modification pattern on the internalization of water soluble β-(1 → 3)-(1 → 4)-glucan by primary murine macrophages

It has been shown that β-(1 → 3)-(1 → 4)-glucans (BG34) from barley and oats can trigger recognition and internalization by murine and human macrophages. Increasing evidence has suggested that macrophage recognition and internalization of BG34 are dramatically affected by the purity of BG34, the molecular weight and chemical modification. In this study, we investigated the structural features of BG34 for macrophage recognition and internalization. We prepared homogeneous BG34s of 10 kDa (BG34-10), 200 kDa (BG34-200) and 500 kDa (BG34-500) with high purity, and then introduced green fluorescence FITC to the reducing ends (Re) or main chain (Mc). The results of size exclusion chromatography, ¹³C NMR, fluorescence microscopy, FACS analyses and MTS assay demonstrated that non-toxic BG34 of 10 kDa (BG34-10) effectively trigger macrophage internalization. The internalization was adversely affected by modifying the main chain of BG34-10 but not the reducing end. Studies using blocking antibodies on several CD11b⁺ and CD11b^? cells suggested that CD11b may play an important role in mediating macrophage internalization of BG34-10. Quantitative RT-PCR and intracellular cytokine stain revealed that macrophages generate increased level of CD11b and TNF-α in response to BG34-10. This study for the first time demonstrated the molecular size (10 kDa) and pattern of modification (reducing end modification) for BG34-10 to mediate macrophage internalization. Since BG34 is water soluble, biocompatible and biodegradable FDA-approved material, this mechanism of BG34-10 can be used to design drug delivery system targeting macrophages.     

Synthesis of β-(1→6)-linked N-acetyl-D-glucosamine oligosaccharide substrates and their hydrolysis by Dispersin B

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Degradation of barley β-glucan by endoglucanase C of Clostridium thermocellum

Summary Endoglucanase C encoded by the celC gene of Clostridium thermocellum has been purified to homogeneity from a recombinant Escherichia coli strain. It was found that this enzyme is highly efficient in degrading glucans with alternating -1,4- and -1,3-linkages but lacks activity on unmodified cellulosic substrates. The properties of endoglucanase C were compared to those of Bacillus subtilis -glucanase, an enzyme used in the brewing industry for -glucan degradation. Both enzyme cause a rapid decrease of the viscosity of barley -glucan as a result of internal chain cleavage. Endoglucanase C hydrolyses non-specifically -1,3- and -1,4-bonds adjacent to unsubstituted or 4-O-substituted cellobiose units. Due to its lower pH optimum and increased thermostability endoglucanase C compares favourably with B. subtilis -glucanase and seems suitable for use in the mashing process of beer brewing.     

The formation of a β-(1→4)-d-galactan chain catalysed by a Phaseolus aureus enzyme

With a particulate enzyme preparation from Phaseolus aureus hypocotyls, UDP-alpha-d-[U-(14)C]galactose served as a precursor for a number of products. One of these products was characterized as a beta-(1-->4)-linked galactan. The ADP-, GDP-, TDP- and CDP- derivatives of alpha-d-galactose did not serve as biosynthetic precursors for any products insoluble in 70% ethanol, nor as substrates for a sugar nucleotide 4-epimerase which is present in the particulate enzyme preparation. The (14)C-labelled beta-(1-->4)-galactan is alkali-insoluble and was characterized by analysis of partial acetolysis products. The labelling pattern of the [(14)C]oligosaccharides derived from acetolysis indicates that (1) only slightly more than two [(14)C]galactose moieties are added to the growing polysaccharide chain on average, and (2) these additions take place at the reducing end of the polysaccharide chain. The radioactive beta-(1-->4)-linked galactan chain represented 8.5% of the radioactivity initially added, and 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. Total hydrolysis of the alkali-insoluble fraction of Phaseolus aureus hypocotyl yielded d-glucose and d-mannose in a 5:1 ratio but no detectable quantities of d-galactose. A trace quantity of a radioactive disaccharide, identified as (1-->3)-linked galactobiose, was isolated from the partial acetolysate of the alkali-insoluble [(14)C]polysaccharide material. Also isolated from this partial acetolysate was a C-1 derivative of [(14)C]galactose, which could not be identified. An alkali-soluble galactose-containing polysaccharide was also synthesized in this enzymic reaction, and represented 20% of the water- and butanol-insoluble products derived from UDP-alpha-d-[(14)C]galactose. The spectrum of radioactive oligosaccharides produced by partial acetolysis of this alkali-soluble polysaccharide material was different from that obtained from the alkali-insoluble polysaccharide, indicating a different structure.     

Biosynthesis of a β-(1 → 4)-mannan in fenugreek seedlings

A particulate enzymatic preparation, extracted from fenugreek seedlings (Trigonella foenum-graecum) catalyses the transfer of mannose from guanosine diphosphate-[U-¹⁴C]mannose and its incorporation into an alkali-soluble polysaccharide. Chemical and enzymatic study of this polysaccharide reveals the presence of only one type of osidic linkage, namely β-(1 → 4)-s-mannopyranosyl. The influence of some factors on this biosynthesis was studied, as well as the MW of the polysaccharide and the existence of an endogenous acceptor.     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

Summary Most cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to -(16) and -(14)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that -(14)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, -(16)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of -(14)-D-galactan epitopes are highly regulated in developing flax roots and that different -linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Effect of senescence and hormone treatment on the activity of a β-1,3-glucan hydrolase in Nicotiana glutinosa leaves

Summary A high level of activity of a -1,3-glucan hydrolase is present in leaves of Nicotiana glutinosa and the enzyme is also present in the roots, midribs, petioles and stems. By comparison, very low levels of -1,4-glucan hydrolase are found throughout the plant. The activity of the -1,3-glucan hydrolase in leaves aged on the plant was found to increase 14-fold during the course of leaf senescence and to reach a maximum in yellow-green leaves. Detached leaves and leaf discs floated on water in the dark showed similar patterns of change.The increase in -1,3-glucan hydrolase activity during senescence is apparently not due to the loss of an inhibitor from young green leaves or to the formation of an enzyme activator in yellow leaves. The enzyme in yellow leaves was electrophoretically indistinguishable from that in green leaves. The hydrolase is not firmly attached to the cell walls and is not present in the particulate fraction sedimenting at 105400xg for 60 min. Within the leaf cell it is therefore likely to be located either in the cytoplasm or in an easily disrupted structure such as a vacuole.The relationship of the hydrolase to leaf senescence was investigated by examining the effect of plant hormones on the changes in level of hydrolase, protein and chlorophyll in leaf discs during senescence. IAA (10 M) and GA₃ (50 M) did not alter the normal patterns of change, whilst Kin (50 M) delayed the loss of protein and chlorophyll and also delayed and decreased the rise in hydrolase activity. In contrast, ABA (190 M) which increased the rate of loss of protein and chlorophyll, also caused a decrease in the rate and extent of the rise in hydrolase.Possible functions of the hydrolase in the leaf are discussed.Abbreviations used throughout text CM-pachyman carboxymethyl pachyman - CM-cellulose carboxymethyl cellulose - BSA bovine serum albumin - ABA abscisic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - Kin kinetin     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1–>6)-β-d-Glcp-(1–>3)-β-d-Glcp-(1–>3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1–>4)-β-d-Glcp-(1–>3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1–>3)-d-Glcp at subsites −2 and −1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases.     

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state. Received: 7 January 1997 / Accepted: 11 February 1997