{beta}-1,4-Glucan occurring in homogenate of Phaseolus aureus seedlings. Possible nascent stage of cellulose biosynthesis in vivo

A small amount of cytoplasmic ß-1,4-glucan, whichmight be involved in the synthesis of cellulose in the cellwall, was found in the homogenate prepared from the hypocotylsof seedlings of Phaseolus aureus. Upon hydrolysis by cellulaseof the 20,000?g pellet from the cytoplasmic fraction of segmentsincubated in a [¹⁴C]-glucose solution, [¹⁴C]-cellobiose wasproduced, with specific radioactivities 3 to 10 times greaterthan those of the cellobiose from cellulose in the cell wallat various incubation periods. The incoporation of radioactivityfrom [¹⁴C]-glucose into this cytoplasmic ß-1,4-glucanwas therefore faster than that into cellulose constituting thecell wall. Hence, it seemed that the former ß-1,4-glucancould be turned over. To examine whether the- cytoplasmic ß-1,4-glucanis carried by some subcellular components, cytoplasmic ß-1,4-glucanin the cell was fractionated by differential centrifugation,two enzyme activities being measured as the markers of subcellularcomponents. The distribution of ß-1,4-glucan was similarto that of UDPG-glucosyltransferase activity but not to thatof IDP-ase activity. The result suggests that the cytoplasmicß-1,4-glucan has some relation to plasma membranes. Coumarin, known as a specific inhibitor for the biosynthesisof cellulose in plant cells, was shown to inhibit the incorporationof radiocarbon from [¹⁴C]-glucose into cytoplasmic ß-1,4-glucanto the same extent as that into cellulose in the cell wall ofthe hypocotyls. ¹ Present address: Department of Biological Science, TohokuUniversity, Kawauchi, Sendai 980, Japan. (Received May 31, 1976; )     

Biosynthesis of Xyloglucan in Suspension-cultured Soybean Cells. Evidence that the Enzyme System of Xyloglucan Synthesis does not Contain {beta}-1,4-Glucan 4-{beta}-D-Glucosyltransferase Activity (EC 2.4.1.12)

Xyloglucan 4-ß-D-glucosyltransferase, an enzyme responsiblefor the formation of the xyloglucan backbone, in a particulatepreparation of soybean cells has been compared with ß-1,4-glucan4-ß-D-glucosyltransferase of the same origin. Thefollowing observations indicate that the enzyme system of xyloglucansynthesis does not contain ß-1,4-glucan 4-ß-D-glucosyltransferaseactivity, although both enzymes transfer the glucosyl residuefrom UDP-glucose to form the ß-1,4-glucosidic linkage:1. The incorporation of [¹⁴C]glucose into xyloglucan dependedon the presence of UDP-xylose in the incubation mixture. 2.No measurable amount of radioactivity was incorporated fromUDP-[¹⁴C]xylose into the cello-oligosaccharides, although theincorporation of [¹⁴C]xylose into xyloglucan depended on thepresence of UDP-glucose in the incubation mixture (Hayashi andMatsuda 1981b). 3. The activity of xyloglucan 4-ß-D-glucosyltransferasewas stimulated more strongly by Mn²⁺ than by Mg²⁺, whereas Mg²⁺was the most active stimulator for the activity of ß-1,4-glucan4-ß-D-glucosyltransferase. 4. An addition of GDP-glucose(100 µM) to the incubation mixture inhibited the activityof xyloglucan 4-ß-D-glucosyltransferase by 17%, whereasthe activity of ß-1,4-glucan 4-ß-D-glucosyltransferasewas inhibited 56% under the same conditions. 5. Irpex exo-cellulasedid not hydrolyze the xyloglucan synthesized in vitro. 6. Theß-1,4-glucan synthesized in vitro was not a branchedxyloglucan because it gave no 2,3-di-O-methyl glucose derivativeon methylation analysis. 7. Pulse-chase experiments indicatedthat the ß-1,4-glucan was not transformed into thexyloglucan. The subcellular distribution of the xyloglucan synthase, however,was similar to that of the ß-1,4-glucan synthase (Golgi-located1,4-ß-D-glucan 4-ß-D-glucosyltransferase).Thus, it appears that the latter enzyme is located at a siteclose to xyloglucan synthase and is set aside for the assemblyof these polysaccharides into the plant cell surface. (Received May 21, 1981; Accepted October 13, 1981)     

Evidence for Several Galactan Synthases in Flax (Linum usitassimum L.) Suspension-Cultured Cells

A membrane fraction from flax cells was able to incorporate[¹⁴C]galactose from UDP-D-[¹⁴C]galactose in vitro. The productsof the reaction, characterized by methylation analysis, consistedof a rß-1,4-galactan (solubilized mainly in water)and a rß-1,3- rß-1,6-galactan (solubilizedmainly in alkali). These results indicated the presence of severalgalactan synthase complexes, as did a profile of the relationshipbetween pH and activity which revealed both a maximum at pH6.5 and a shoulder at pH 8. Moreover, galactan synthase activitieswere found at two densities: 1.125 g cm^–3 (Golgi membranes)and 1.07–1.08 g cm^–3 (corresponding to low-densityvesicles). Partial characterization of one enzymatic system (maximaly activeat pH 8 in the presence of 5 mM MgCl₂) was achieved. The K_mfor UDP-galactose and V_max were 38 µM and 4.5 nmol h^–1(mg protein)^–1, respectively. (Received June 6, 1993; Accepted September 22, 1993)     

Effects of Culture Conditions on Cell Division and Composition of Regenerated Cell Walls in Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere grown in a liquid medium, and the effects of osmolarityand growth regulators on cell division and on the compositionof regenerated cell walls were investigated. The concentrationof mannitol optimal for cell division was 0.3–0.4 M. Thepresence of 2,4-D was essential for cell division, and 6-benzylaminopurine(BAP) enhanced cell division at concentrations of 0.03–0.1ppm. However, the composition of regenerated cell walls wasabnormal under suspension culture; the predominant sugar wasglucose, indicating that the regenerated cell walls consistedmostly of glucans, and that the other cell-wall components werereleased into the medium. Mannitol, 2,4-D, and BAP at variousconcentrations did not significantly affect the sugar compositionof the regenerated cell walls. Compared with liquid culture,cell division was stimulated when protoplasts were culturedon agar, and their regenerated cell walls had a compositionsimilar to that of the original culture. The importance of thephysical environment for the deposition of polysaccharide componentsin cell walls and the interrelationship between cell divisionand the regeneration of cell walls by protoplasts are discussed. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan. (Received June 10, 1981; Accepted December 12, 1981)     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

Induction of chitinase and {beta}-1,3-glucanase activity in sunflower suspension cells in response to an elicitor from Phytophthora megasperma f. sp. glycinea (Pmg). Evidence for regulation by ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC)

In heterotrophic cell suspensions of sunflower (Helianthus annuusL. cv. Spanners Allzweck) the effect of Pmg elicitor, a fungalelicitor preparation from Phytophthora megasperma f. sp. glycinea,on the induction of chitinase and ß-1,3-glucanaseactivity was studied in relation to changes in ethylene biosynthesis.Dose-response experiments with Pmg elicitor showed that theonset of the induction of intracellular chitinase and ß-1,3-glucanaseactivity coincided or followed a transient rise in ethyleneand particularly endogenous 1-aminocyclopropane-1-carboxylicacid (ACC) levels within 5 h of application. Treatment with5 µg ml^–1 elicitor stimulated ethylene and ACC levels1.6-fold and 4-fold, relative to control, respectively. Themolar ratio of ACC to ethylene changed from approximately 3:1in controls to 9:1 in treated cells. During further incubation,ethylene formation and, to a lesser degree, ACC levels declinedand the ACC/ethylene ratio increased to 56:1 in elicitor-treatedcells. On a protein basis, the activities of ß-1,3-glucanaseand chitinase increased approximately 5-fold and 8-fold, respectively,48 h after elicitor application. Additional treatment with theACC synthesis inhibitor aminoethoxyvinyiglycine (AVG) decreasedelicitor-induced enzyme activities and the levels of both ethyleneand ACC. Elicitor effects on chitinase and ß-1,3-glucanaseactivities could be fully restored when ACC was additionallyapplied. Concomitantly, the ACC/ ethylene ratio increased. Neithertreatments with ACC alone, which simultaneously increased internalACC and ethylene levels, nor treatments with AVG alone, whichsimultaneously reduced ACC and ethylene levels, could generallystimulate chitinase or ß-1,3-glucanase activitiesin the cells. It is suggested that ACC functions as a promotingfactor in the induction of chitinase and ß-1,3-glucanaseactivity triggered by Pmg elicitor and appears to reverse aninhibiting influence of ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid, chitinase, ß-1,3-glucanase, ethylene, Helianthus cellsuspension cultures, Phytophthora megasperma-elicitor     

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal ß-Gal residueswas investigated by screening sulfotransferase activity presentin 37 human tissue specimens toward the following synthesizedacceptor moieties: Galß1,3GalNAc-O-Al, Galß1,4GlcNAcß-O-Al,Galß1,3GlcNAcß-O-Al, and mucin-type Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnstructures containing a C-3 methyl substituent on either Gal.Two distinct types of Gal: 3-O-sulfotransferases were revealed.One (Group A) was specific for the Galß1, 3GalNAc-linkage and the other (Group B) was directed toward the Galß1,4GlcNAcbranch ß1,6 linked to the blood group T hapten. Enzymeactivities found in breast tissues were unique in showing astrict specificity for the T-hapten. Galß-O-allylor benzyl did not serve as acceptors for Group A but were veryactive with Group B. An exainination of activity present insix human sera revealed a specificity of the serum enzyme towardß1,3 linked Gal, particularly, the T-hapten withoutß1,6 branching. Group A was highly active toward T-haptenlacrylamidecopolymer, anti-freeze glycoprotein, and fetuin O-glycosidicasialo glycopeptide; less active toward fetuin triantennaryasialo glycopeptide; and least active toward bovine IgG diantennaryglycopeptide. Group B was moderately and highly active, respectively,with the latter two glycopeptides noted and least active withthe first two. Competition experiments performed with Galß1,3GaLNAc-O-Aland Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnhaving a C-3 substituent (methyl or sulfate) on either Gal reinforcedearlier findings on the specificity characteristics of GroupA and Group B. Group A displayed a wider range of optimal activity(pH 6.0–7.4), whereas Group B possessed a peak of activityat pH 7.2. Mg²⁺ stimulated Group A 55% and Group B 150%, whereasMn⁺² stimulated Group B 130% but inhibited Group A 75%. Ca²⁺stimulated Group B 100% but inhibited Group A 35%. Group A andGroup B enzymes appeared to be of the same molecular size (<100,000Da) as observed by Sephacryl S-100 HR column chromatography.The following effects upon Gal: 3-O- sulfotransferase activitiesby fucose, sulfate, and other substituents on the carbohydratechains were noted. (1) A methyl or GlcNAc substituent on C-6of GalNAc diminished the ability of Galß1,3GalNAc-O-Alto act as an acceptor for Group A. (2) An 1,3-fucosyl residueon the ß1,6 branch in the mucin core structure didnot affect the activity of Group A toward Gal linked ß1,3to GalNAc-. (3) Lewis x and Lewis a terminals did not serveas acceptors for either Group A or B enzymes. (4) Eliminationof Group B activity on Gal in the ß1,6 branch owingto the presence of a 3-fucosyl or 6-sulfo group on GlcNAc didnot hinder any action toward Gal linked ß1,3 to GalNAc.(5) Group A activity on Gal linked ß1,3 to GalNAcremained imaffected by 3'-sulfation of the ß1,6 branch.The reverse was true for Group B. (6) The acceptor activityof the T-hapten was increased somewhat upon C-6 sulfation ofGalNAc, whereas, C-6 slalylation resulted in an 85% loss ofactivity. (7) A novel finding was that Galß1,4GlcNAcß-O-Aland Galß1,3GlcNAcß-O-M, upon C-6 sulfationof the GlcNAc moiety, became 100% inactive and 5- to 7-foldactive, respectively, in their ability to serve as acceptorsfor Group B. human tissues glycoprotein galactose:sulfotransferase specificities kinetic properties     

Xyloglucan and r{beta}-D-Glucan in Cell Walls of Rice Seedlings

Cell walls of 4-day old rice seedlings were extracted successivelywith ammonium oxalate-oxalic acid, 4% KOH and 24% KOH. A rß-D-glucanpreparation and a xyloglucan preparation were isolated fromthe 4% KOH extract and 24% KOH extract, respectively. Methylationanalysis and enzymic degradation studies of the polysaccharidesshowed that the former was built up predominantly of repeating-oligosaccharideunits of 3-O-rß-cellobiosyl-D-glucose and 3-O-rß-cellotriosyl-D-glucosein a molar ratio of 2.6 : 1.0, and the latter was of repeating-oligosaccharideunits of -D-xylosyl-(16)-rß-D-glucosyl-(14)-[-D-xylosyl-(16)]-rß-D-glucosyl-(14)-D-glucose,-D-xylosyl-(16)-rß-D-glucosyl-(14)-D-glucose and cellobiose. ¹ Present address: Department of Botany, Iowa State University,Ames, Iowa 50011, U.S.A. (Received August 29, 1981; Accepted January 12, 1982)     

{beta}1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay

Lymphocytic ß1,4-galactosyltransferase (ß1,4-GalTase,EC 2.4.1.38 [EC] ) activity was measured in B cells using a neoglycoprotein,N-acetylglucosamine-phenylisothlocyanate-bovine serum albumin(GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linkedimmunosorbent assay (ELISA)-based method. This assay provedto be much simpler to use than the lengthy and expensive radiochemicalassays commonly used, and has the additional advantage thatit specifically detects the enzyme mediating transfer via theGalß1,4GlcNAc linkage. A F(ab')₂ antibody againstGalTase was able to specifically inhibit the reaction. Greatersensitivity for ß1,4-GalTase activity was obtainedusing GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin.Low levels of ß-galactosidase activity were detectablein lymphocyte cell lysates at acidic pH, although such activitywas not detectable at the neutral pH used in the ß1,4-GalTaseactivity assay. Using this assay with the GlcNAc-pITC-BSA acceptor,similar ß1,4-GalTase activities were observed in CD19⁺B cells from patients with rheumatoid arthritis (RA) to thoseseen in normal control individuals. ELISA ß1,4-galactosyltransferase lymphocyte neoglycoprotein radiochemical     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-{beta}-glucosides using UDP gal:GlcNAc {beta}1,4 galactosyltransferase

We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside G_M3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =6–9). All were labeled with ³H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors     

Purification and Properties of an Extracellular Endo-1,4-r{beta}-Glucanase from Suspension-Cultured Poplar Cells

An endo-1,4-rß-glucanase (EC 3.2.1.4 [EC] ) was purifiedto apparent homogeneity from the culture medium of poplar (Populusalba L.) cells by sequential anion-exchange, hydrophobic, andgel-filtration chromatography. The preparation of extracellularrß-glucanase was homogeneous on SDS-polyacrylamidegel electrophoresis (PAGE) and native PAGE. The molecular weight,as determined by SDS-PAGE was 50,000, whereas that determinedby gel filtration was 40,000. The isoelectric point (pI) was5.5. The purified enzyme catalyzed the endohydrolysis of carboxy-methylcellulosewith a pH optimum of 6.0 and a k_m of 1.0 mg ml^–1. Theenzyme specifically cleaved the 1,4-rß-glucosyl linkagesof carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan,although the last was hydrolyzed more slowly than the othertested substrates. The activity of the endo-1,4-rß-glucanaseincreased up to the early stage of the mid-logarithmic phaseof growth and then decreased rapidly, suggesting that the rß-glucanaseis induced before cell development. (Received April 28, 1993; Accepted July 19, 1993)     

Levels of {beta}-Glucan and Lignin in Elongating Internodes of Deepwater Rice

Partial submergence or treatment with either ethylene or gibberellicacid (GA₃ induces rapid growth in deepwater rice (Oryza sativaL.). We correlated the synthesis of two cell wall componentswith two phases of internodal elongation, namely (13,14)-ß-glucanformation with cell elongation and lignification with differentiationof the secondary cell wall and cessation of growth. The contentof ß-glucan was highest in the zone of cell elongationin internodes of air-grown plants and plants that were inducedto grow rapidly by submergence. In the intercalary meristemand in the differentiation zone of the internode, ß-glucanlevels were ca. 70% lower than in the zone of cell elongation.The outer cell layers, enriched in epidermis, contained moreß-glucan in submerged, rapidly growing internodesthan in air-grown, control internodes. The ß-glucancontent of the inner, parenchymal tissue was unaffected or slightlylowered by submergence. The epidermis appears to be the growth-limitingstructure of rapidly growing rice internodes. We hypothesizethat elevated levels of ß-glucan contribute to elongationgrowth by increasing the extensibility of the cell wall. Lignificationwas monitored by measuring the content of lignin and the activitiesof two enzymes of the lignin biosynthetic pathway, coniferylalcohol dehydrogenase (CAD) and phenylalanine ammonia-lyase(PAL), in growing and non-growing regions of the internode.Using submerged whole plants and GA₃-treated excised stem segments,we showed that lignin content and CAD activity were up to sixfoldlower in newly formed internodal tissue of rapidly growing ricethan in slowly growing tissue. No differences were observedin parts of the internode that had been formed prior to inductionof growth. PAL activity was reduced throughout the internodeof submerged plants. We conclude that lignification is one ofthe processes that is suppressed to permit rapid growth. ¹ This work was supported by the National Science Foundationthrough grants No. DCB-8718873 and DCB-9103747 and by the Departmentof Energy through grant No. DE-FGO2-90ER20021. M.S. was therecipient of a fellowship from the Max Kade Foundation.     

Herbicide-tolerant Transgenic Pineapple (Ananas comosus) Produced by Microprojectile Bombardment

Transformed plants of the commercially important Thai pineapple(Ananas comosus‘Phuket’) were produced followingmicroprojectile-mediated delivery of the plasmid AHC25, carryingthe ß-glucuronidase (gus) reporter gene and the bialaphosresistance (bar) gene for herbicide tolerance, into leaves ofmicropropagated shoots. Transformed plants were regeneratedfrom bombarded leaf bases on Murashige and Skoog-based mediumcontaining 0.5 mg l^-12,4-dichlorophenoxyacetic acid, 2.0 mgl^-16-benzylamino purine and 0.5 mg l^-1phosphinothricin. Integrationand expression of thebar gene in regenerated plants was confirmedby Southern analysis and RT-PCR, respectively. Regenerated plantswere assessed in vitro and under glasshouse conditions for theirtolerance to the commercial herbicide Basta^TM, containing glufosinateammonium as the active component. Plants sprayed with Basta^TMcontainingconcentrations of glufosinate ammonium up to 1400 mg l^-1remainedhealthy and retained their pigmentation. The generation of herbicide-tolerantpineapple will facilitate more efficient weed control in thiswidely cultivated tropical crop. Copyright 2001 Annals of BotanyCompany bar gene, Biolistics, herbicide tolerance, pineapple, phosphinothricin (PPT)     

Comparative Studies of Acid Glycosidases from Three Legumes

The major isoenzymes of -mannosidase (EC 3.2.1.24 [EC] ) and ß-galactosidase(ECf 3.2.1.23 [EC] ) have been separated from cotyledons of gardenpea, Pisum sativum L. (Vicieae), chick pea, Cicer arietinumL. (Cicereae), and cowpea, Vigna unguiculata (L.) Walp. (Phaseoleae).Some of their properties have been determined, including pHoptima, K_m values for p-nitrophenyl glycosidc substrates, andthe effects of several inhibitors. Swainsonine, an indolizidinealkaloid, was the most effective inhibitor of mannosidase 1,with I₃₀ values of 5.6 x 10^–8 M (cowpea), 1x 10^–7M (chick pea) and 2.9 x 19^–7 M (pea). The most effectiveinhibitor of ß-galactosidase 2 from all sources wasD-galactonic acid-1,4-lactonwe (-lactone), with K_i values rangingbetween 3.0 and 3.9x 10^–3 M. An inhibitor of the E. coliß-galactosidose, p-aminophenyl thio-ß-D-galactopyranoside,did not inhibit any of the legume ß-galctosidases;rather it enhanced the activites of the enzymes from chick peaand cowpea cotyledons. Etiolated hull and seed tissues frompea pods developing in darkness contained similar acid glycosidaseactivities to normal green tissues, thus the chloroplast isan unlikely location for ß-galactosidase 2. The majorß-galactosidasesdetected with an indigogenic substrate (5-bromo-4-chloro-3-indoxyl-ß-D-galactopyranoside)following gel electrophoresis of extracts from pea hull, seedcoats and cotyledons appeared to be different from ß-galactosidase2. Acid glycosidase, cotyledon, isoenzyme, -lactone, legume, swainsonine     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

The Occurrence of 1,3-{beta}-Glucanase in Healthy and Verticillium-infected, Resistant and Susceptible Tomato Plants

Leaf, stem, and root extracts of near-isogenic tomato plantscv. Craigella, resistant and susceptible to Verticillium albo-atrum,showed constitutive 1,3-ß-glucanase activity whichincreased following inoculation with the pathogen. Partiallypurified enzyme extracts were obtained by dialysing a 30–80%ammonium sulphate fraction of the tissue brei. The enzyme hadpH and temperature optima of 5?5 and 44 ?C respectively, withhigh activity between 50 and 60 ?C. The response to laminarinconcentration was linear between 1?2 and 7?5 mg ml^–1.Root inoculation of susceptible plants with 10⁶ propagules ml^–1V. albo-atrum led to a umform 300 per cent increase in all steminternodes except the terminal one, which was 500 per cent ofthe controls. No spatial relationship of enzyme activity tothe localization of fungus within the stem was apparent. Petioles,leaves, and roots of susceptible infected plants similarly showedan increase in activity but less than that in stems. Changedlevels of stern enzyme activity at different times after inoculationwere associated with reductions in the number of vessels containinghyphae. Extracts of plants of the resistant isoline showed increasedglucanase activity over controls, but this was substantiallylower than that in susceptible plants and was associated withthe greatly reduced mycelial colonization in resistant plants. It is concluded that single gene resistance in tomato to Verticilliumis not associated with innately higher levels of 1,3-ß-glucanasein healthy plants. The increased activity in infected plantsis proportional to the overall quantity of pathogen in the plantor of pathogenic metabolites.     

Purification and Characterization of Wall-bound Exo-l,3-{beta}-D-Glucanase from Barley (Hordeum vulgare L.) Seedlings

A ß-D-glucanase activity hydrolyzing 1,3:1,4-ß-D-glucanwas released from the cell walls of barley by 3M LiCl treatment.It was purified by sequential cation-exchange, gel-filtrationand hydrophobic chromatography. The molecular mass of the glucanasewas 66 kDa as determined by SDS-polyacrylamide gel electrophoresis.Sequence determination of the first thirty amino acids of theN-terminus revealed a high homology of this enzyme to the Pseudomonasl,4-ß-D-glucosidase (56.5%). The purified ß-D-glucanasehas a pH optimum at 5.0, and hydrolyzes oligosaccharides containingß-D-1,3 or ß-D-1,4 linkage. The glucanaseshowed maximum hydrolytic activity toward laminaritetraose,the rate being about two times that of cellotetraose and aboutfour times that of gentiobiose. Polysaccharides such as lichenan,l,3:l,4-ß-D-glucan (from barley), laminarin and pustulanare also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose,xyloglucan and maltose. The purified ß-D-glucanaseyielded monomeric glucose from laminarihexaose, and exhibitedcharacteristics of an exo-l,3-ß-D-glucanase (EC 3.2.1.58 [EC] ).The activity and biochemical characteristics of this enzymesuggest that it is an exo-l,3-ß-D-glucanase involvedin the rapid turnover of l,3:l,4-ß-D-glucan in barleycell walls during seedling growth. (Received September 24, 1996; Accepted December 9, 1996)     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan