Cellulases Can Enhance beta-Glucan Synthesis.

beta-Glucan synthesis from uridine diphosphoglucose by pea epicotyl tissue slices is increased two- to threefold by preliminary, short-term treatment with cellulases purified from auxin-treated peas. We suggest that cellulases introduce chain ends in accessible regions of cellulose microfibrils which then act as primers for chain elongation.

Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase.

BACKGROUND: Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases. RESULTS: . The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis. CONCLUSIONS: The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.

Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase.

BACKGROUND: Barley beta-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta-D-glucans and beta-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. RESULTS: When conduritol B epoxide and 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Odelta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I), 4(III), 4(V)-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the -1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 A) with the Oepsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C(1) conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. CONCLUSIONS: X-ray crystallography is used to define the three key intermediates during catalysis by beta-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex.

Stereochemical course of glucan hydrolysis by barley (1-->3)- and (1-->3, 1-->4)-beta-glucanases.

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1-->3, 1-->4)-beta-glucan by barley (1-->3)-beta-glucanase (E.C. 3.2.1.39) isoenzyme GII and (1-->3, 1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by 1H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e-->e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of beta-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

Crystallization and preliminary X-ray analysis of (1,3)- and (1,3;1,4)-beta-D-glucanases from germinating barley.

(1,3)-beta-D-Glucanase isoenzyme GII and (1,3;1,4)-beta-D-glucanase isoenzyme EII from barley have been crystallized by the hanging drop method in the presence of ammonium sulphate. The crystals of the (1,3)-beta-D-glucanase, which diffract to about 1.8 A resolution, belong to the trigonal space group P3(1)2(1)2 (or P3(2)2(1)2) with cell constants a = b = 86.9 A, c = 156.0 A and contain two molecules in the asymmetric unit. The crystals of the (1,3;1,4)-beta-D-glucanase which diffract to better than 1.8 A resolution, belong to the tetragonal space group P4(3)2(1)2 (or P4(1)2(1)2) with cell constants a = b = 87.4 A, c = 109.5 A and contain one molecule in the asymmetric unit.

Evolution and differential expression of the (1-->3)-beta-glucan endohydrolase-encoding gene family in barley, Hordeum vulgare.

The (1-->3)-beta-D-glucan glucanohydrolases [(1-->3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1-->3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1-->3,1-->4)-beta-glucan 4-glucanohydrolases [(1-->3,1-->4)-GGH; EC 3.2.1.73] further indicate that the (1-->3)-GGHs and (1-->3,1-->4)-GGHs are derived from a single 'super' gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1-->3,1-->4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1-->3,1-->4)-beta-glucans that are the major constituents in barley walls, while the (1-->3)-GGHs, which are unable to degrade the plant (1-->3,1-->4)-beta-glucans, can hydrolyse the (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls.

Barley (1----3,1----4)-beta-glucanase isoenzyme EI gene expression is mediated by auxin and gibberellic acid.

Treatment of young barley leaves with indole acetic acid (IAA) or gibberellic acid (GA3) results in a dramatic increase in levels of (1----3,1----4)-beta-glucanase isoenzyme EI transcripts. In young roots of comparable age, levels of isoenzyme EI mRNA are high; IAA inhibits expression while GA3 has no effect on mRNA levels. The addition of both abscisic acid and GA3 to leaves, roots and aleurone layers leads to higher levels of (1----3,1----4)-beta-glucanase isoenzyme EI mRNA than is found with Ga3 alone. Little or no expression of (1----3,1----4)-beta-glucanase isoenzyme EII is detected in vegetative tissues, but in isolated aleurone layers GA3 enhances levels of isoenzyme EII transcripts, as does IAA. Thus, the two barley (1----3,1----4)-beta-glucanase genes respond quite differently to phytohormone treatment, depending on the tissue and its stage of development.

Differences in the thermostabilities of barley (1----3,1----4)-beta-glucanases are only partly determined by N-glycosylation.

Barley (1----3,1----4)-beta-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzyme EII carries 4% by weight carbohydrate and is more stable at elevated temperatures than isoenzyme EI, which has no associated carbohydrate. The relationship between carbohydrate content and thermostability has been investigated by treatment of the two isoenzymes with N-glycopeptidase F (EC 3.5.1.52). Removal of carbohydrate from isoenzyme EII results in a decrease in the enzyme's thermostability, but treatment of isoenzyme EI with the N-glycopeptidase F has no effect. In addition, removal of a single N-glycosylation site in isoenzyme EII (Asn190-Ala-Ser) by site-directed mutagenesis of the corresponding cDNA led to a reduction in thermostability, while the introduction of this site into isoenzyme EI enhanced stability. We conclude that N-glycosylation of Asn190 enhances the stability of isoenzyme EII at elevated temperatures, but that other factors related to their primary structures also contribute to the differences in thermostabilities of the barley (1----3,1----4)-beta-glucanases.

Heterologous expression of cDNAs encoding barley (Hordeum vulgare) (1-->3)-beta-glucanase isoenzyme GV.

Two cDNAs have been isolated from libraries generated from poly(A)+RNA of young barley roots and leaves, using a cDNA encoding barley (1-->3)-beta-glucanase isoenzyme GII as a probe. Nucleotide sequence analyses and ribonuclease protection assays show that the two cDNAs differ only in the length of their 3'-untranslated regions; the corresponding mRNAs are likely to originate from a single gene by tissue-specific processing at separate polyadenylation sites. When the coding region of the cDNA is expressed in E. coli, the resultant protein catalyses the hydrolysis of (1-->3)-beta-glucan with an action pattern characteristic of a (1-->3)-beta-glucan endohydrolase (EC 3.2.1.39). The enzyme has been designated isoenzyme GV of the barley (1-->3)-beta-glucanase family).

Post-translational processing of barley beta-glucan endohydrolases in the baculovirus-insect cell expression system.

Two cDNAs encoding barley (1-->3,1-->4)-beta-glucanase (EC 3.2.1.73) isoenzymes EI and EII have been expressed in Spodoptera frugiperda (Sf9) cell cultures using the baculovirus AcNPV vector. Modifications to both the 5' and 3' ends of the cDNAs were required before satisfactory levels of expression were obtained. The modified cDNAs directed high levels of (1-->3,1-->4)-beta-glucanase expression in the Sf9 insect cell cultures, with yields of approximately 10 mg/liter of isoenzyme EI (expEI) and 15 mg/liter of isoenzyme EII (expEII). Amino acid sequence analyses showed that the expressed enzymes were processed correctly at their amino termini. However, affinity chromatography of the isoenzyme expEII on concanavalin-A (conA)-Sepharose indicated that, although the enzyme is glycosylated, the structures of the carbohydrate chains differ from those of the native enzyme. When a cDNA encoding the homologous barley (1-->3)-beta-glucanase (EC 3.2.1.39) isoenzyme GII was expressed in insect cells, aberrant amino-terminal processing of the nascent polypeptide was sometimes observed. The forms with incompletely removed signal peptides retained their substrate specificity, but exhibited slightly reduced catalytic efficiency, altered chromatographic behavior, and reduced stability at elevated temperatures. The results show that high levels of expression of recombinant plant proteins can be obtained in insect cells, but they emphasize the need to characterize thoroughly the products that are expressed in the heterologous insect cell system before comparisons are made with the native enzyme or with engineered enzyme mutants.

Purification and characterization of a (1-->3)-beta-D-glucan endohydrolase from rice (Oryza sativa) bran.

A (1-->3)-beta-glucanase with an apparent M(r) of 29,000 and an isoelectric point of 4.0 has been purified 2000-fold from extracts of rice bran, using fractional precipitation with ammonium sulfate, anion exchange chromatography, size-exclusion chromatography, chromatofocussing, and hydrophobic interaction chromatography. The enzyme can be classified with the EC 3.2.1.39 group, because it releases laminarabiose and higher laminara-oligosaccharides from linear (1-->3)-beta-D-glucans with an action pattern that is typical of (1-->3)-beta-D-glucan endohydrolases. However, the introduction of substituents or branching in the (1-->3)-beta-D-glucan substrates causes a marked decrease in the rate of hydrolysis. Thus, substituted or branched (1-->3)-beta-D-glucans of the kind commonly found in fungal cell walls are less susceptible to hydrolysis than essentially linear (1-->3)-beta-D-glucans. Kinetic analyses indicate an apparent Km of 42 microM, a kcat constant of 67 s-1, and a pH optimum of 5.0 during hydrolysis of the (1-->3)-beta-D-glucan, laminaran, from Laminaria digitata. The first 60 NH2-terminal amino acid residues of the purified rice (1-->3)-beta-glucanase contain blocks of amino acids that are conserved in other cereal (1-->3)-beta-glucanases. Although the precise tissue location and function of the enzyme in rice bran are not known, it is likely that it is concentrated in the aleurone layer and that it plays a preemptive role in the protection of ungerminated grain against pathogen attack.

The sequence statistics and solution conformation of a barley (1----3, 1----4)-beta-D-glucan.

The sequence statistics and aqueous solution conformation of the 40 degrees water-soluble (1----3,1----4)-beta-D-glucan isolated from barley (Hordeum vulgare) have been modeled realistically using the known sequence-distributions of (1----3) and (1----4) linkages, theoretical conformational analysis, and the statistical mechanical theory of polymer-chain conformation. This barley beta-glucan fraction consists of (1----4)-beta-glucooligosaccharides, predominantly of d.p. 4 or less, joined by single beta-(1----3) linkages. Approximate treatments of the sequence statistics which do not take into account the small mole fraction (approximately 2%) of (1----4)-beta-glucooligosaccharides of d.p. approximately 10 significantly underestimate the chain extension in solution. A correct prediction of the observed chain extension is achieved when these longer, highly extended (1----4)-beta-glucooligosaccharide blocks are included in a model which randomly incorporates all (1----4)-beta-glucooligosaccharide segments in the proportions observed experimentally. Chain flexibility in the 40 degrees water-soluble beta-glucan fraction is shown to arise principally from the isolated beta-(1----3) linkages; blocks of two or more contiguous beta-(1----3) linkages provide a source of additional flexibility which may influence the properties of barley beta-glucan fractions containing a significant proportion of such sequences.

Polysaccharide hydrolases in germinated barley and their role in the depolymerization of plant and fungal cell walls.

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (1 --> 3,1 --> 4)-beta-glucans which comprise in excess of 90% by weight of these walls. The (1 --> 3,1 --> 4)-beta-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (1 --> 3,1 --> 4)-beta-glucans, but beta-glucan exohydrolases and beta-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (1 --> 3,1 --> 4)-beta-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 --> 3)-beta-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 --> 4)-beta-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 --> 4)-beta-xylan exohydrolases and alpha-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed.

Purification and properties of three (1-->3)-beta-D-glucanase isoenzymes from young leaves of barley (Hordeum vulgare).

Three (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1-->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1-->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1-->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1-->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1-->3;1-->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1-->3;1-->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1-->3;1-->4)-beta-D-glucans, (1-->6)-beta-D-glucan, CM-cellulose, insoluble (1-->3)-beta-D-glucans or aryl beta-D-glycosides.

Molecular evolution of plant beta-glucan endohydrolases.

The evolutionary relationships of two classes of plant beta-glucan endohydrolases have been examined by comparison of their substrate specificities, their three-dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1-->3)-beta-glucanases and (1-->3,1-->4)-beta-glucanases from higher plants share a common ancestry and, in all likelihood, that the (1-->3,1-->4)-beta-glucanases diverged from the (1-->3)-beta-glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1-->3,1-->4)-beta-glucanases from (1-->3)-beta-glucanases does not appear to have invoked 'modular' mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate-binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine-tuning of substrate specificity. The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1-->3)-beta-Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1-->3)-beta-glucans of fungal cell walls, appear to have been recruited to generate (1-->3,1-->4)-beta-glucanases, which quite specifically hydrolyse plant cell wall (1-->3,1-->4)-beta-glucans in the graminaceous monocotyledons during normal wall metabolism. Thus, one class of beta-glucan endohydrolase can degrade beta-glucans in fungal walls, while the other hydrolyses structurally distinct beta-glucans of plant cell walls. Detailed information on the three-dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate-binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.

Structure-function relationships of beta-D-glucan endo- and exohydrolases from higher plants.

(1-->3),(1-->4)-beta-D-Glucans represent an important component of cell walls in the Poaceae family of higher plants. A number of glycoside endo- and exohydrolases is required for the depolymerization of (1-->3),(1-->4)-beta-D-glucans in germinated grain or for the partial hydrolysis of the polysaccharide in elongating vegetative tissues. The enzymes include (1-->3),(1-->4)-beta-D-glucan endohydrolases (EC 3.2.1.73), which are classified as family 17 glycoside hydrolases, (1-->4)-beta-D-glucan glucohydrolases (family 1) and beta-D-glucan exohydrolases (family 3). Kinetic analyses of hydrolytic reactions enable the definition of action patterns, the thermodynamics of substrate binding, and the construction of subsite maps. Mechanism-based inhibitors and substrate analogues have been used to study the spatial orientation of the substrate in the active sites of the enzymes, at the atomic level. The inhibitors and substrate analogues also allow us to define the catalytic mechanisms of the enzymes and to identify catalytic amino acid residues. Three-dimensional structures of (1-->3),(1-->4)-beta-D-glucan endohydrolases, (1-->4)-beta-D-glucan glucohydrolases and beta-D-glucan exohydrolases are available or can be reliably modelled from the crystal structures of related enzymes. Substrate analogues have been diffused into crystals for solving of the three-dimensional structures of enzyme-substrate complexes. This information provides valuable insights into potential biological roles of the enzymes in the degradation of the barley (1-->3),(1-->4)-beta-D-glucans during endosperm mobilization and in cell elongation.

Purification and chemical properties of two 1,3;1,4-beta-glucan endohydrolases from germinating barley.

Two 1,3;1,4-beta-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3;1,4-beta-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants.

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide.

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.

Developmental Regulation of (1-->3, 1-->4)-beta-Glucanase Gene Expression in Barley : Tissue-Specific Expression of Individual Isoenzymes.

Two genes encode (1-->3,1-->4)-beta-d-glucan 4-glucanohydrolase (EC 3.2.1.73) isoenzymes EI and EII in barley (Hordeum vulgare L.). Specific DNA probes have been used in Northern analyses to examine the developmental regulation of individual (1-->3,1-->4)-beta-glucanase genes in the aleurone and scutellum of germinated grain and in young leaves and young roots. In aleurone and scutella excised from germinated grain, mRNAs encoding both isoenzymes are present but developmental patterns differ between the two tissues. Thus, levels of both isoenzyme EI and EII mRNA increase significantly in the aleurone between 1 and 3 days after the initiation of germination. In the scutellum, isoenzyme EI mRNA predominates and decreases as germination proceeds. Isoenzyme EI mRNA appears in young leaves approximately 8 days after the initiation of germination and levels rise until about 20 days. Enzyme activity in leaf extracts parallels the development of isoenzyme EI mRNA. No isoenzyme EII mRNA is detected in the leaves in this period. Analysis of RNA from different leaf segments indicates that the isoenzyme EI mRNA is distributed relatively evenly along the length of the leaf. In young roots, mRNA encoding (1-->3,1-->4)-beta-glucanase isoenzyme EI is detected at high levels 3 to 6 days after the initiation of germination; again, little or no isoenzyme EII mRNA is found. Overall, transcription of the (1-->3,1-->4)-beta-glucanase isoenzyme EII gene appears to be restricted to the germinating grain, whereas isoenzyme EI is expressed in a wider range of tissues during seedling development.

Tissue Slice and Particulate beta-Glucan Synthetase Activities from Pisum Epicotyls.

beta-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg(2+) optimum, apparent K(m)) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various beta-glucanases all show that both beta-1,3 and beta-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. beta-1,4 Linkages predominate at low substrate (5 mum) concentration. Kinetic data indicate that the capacity to synthesize beta-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.