Homonojirimycin and N-methyl-homonojirimycin inhibit N-Iinked oligosaccharide processing

Homonojirimycin (HNJ) and N-methylhomonojirimycin (MHNJ) weretested as inhibitors of the purified glycopro-tein processingenzymes, glucosidase I and glucosidase II. MHNJ was a reasonablygood inhibitor of glucosidase I (K₁ = 1 x 10^–6 M) andwas about three times as effective on this enzyme as was HNJ.On the other hand, HNJ inhibited glucosidase II with a K₁ ofabout 1 x 10^x6 M, whereas MHNJ was three times less effective(K₁ = 3 x 10^–5 M). However, the butyl derivative of HNJhad very low activity toward these two processing glucosidases.HNJ and its methyl derivative were also tested in vivo usinginfluenza virus-infected MDCK cells, and measuring the inhibitionof N-linked oligosaccharide processing of the viral envelopeglycoproteins. With 100 µg/ml of MHNJ in the medium, essentiallyall of the N-linked oligosaccharide chains of the virus wereof the "high-mannose" type with the major structure being characterizedas Glc₃Man₉(GlcNAc)₂. Similar results were obtained with HNJalthough this compound was less effective in vivo as well asin vitro. These results are in keeping with these inhibitorsbeing effective at the glucosidase I step. Both inhibitors werealso tested in MDCK cell cultures to determine whether theyaffected the in vivo synthesis of proteins, or of lipid-linkedsaccharides. In contrast to deoxynojirimycin, which has beenreported to inhibit the formation of lipid-linked saccharides,no effects were seen on either the incorporation of mannoseinto lipid-linked saccharides or the incorporation of leucineinto protein. glucosidase lipid influenza virus oligosaccharide     

N-Glycosylation in trypanosomatid protozoa

Trypanosomatid protozoa are parasites of considerable medicaland economic importance in developing countries. The pathwayleading to N-glycosylation in these micro-organisms is characterizedby the following features: (i) dolichols are composed of only10–13 isoprene units; (ii) oligosaccharides transferredin N-glycosylation have the compositions Man_{(6, 7, 9)}GlcNAc₂,depending on the species; (iii) trypanosomatids are unable tosynthesize dolichol-P-Glc and, in addition, some species lackcertain dolichol-P-Man-dependent mannosyltransferases; (iv)the oligosaccharyl-transferase does not require the presenceof glucose units in the oligosaccharide in order to catalysean efficient transfer reaction; (v) trypanosomatids have a glucosidaseII-like enzyme, but lack glucosidase I; (vi) glucosidase IIis required for deglucosylation of oligosaccharides glucosylatedby the UDP-Glc: glycoprotein glucosyltransferase, an activityfirst detected in those parasites; (vii) the structures of polymannose-typecompounds in these protozoa have no significant differenceswith those of their mammallan counterparts except for the presence,in certain species, of oligosaccharides having galactofuranoseunits linked to external mannose residues; (viii) biantennarycomplex-type oligosaccharides having in some cases terminal     

An analysis of seed development in Pisum sativum XIX. Effect of mutant alleles at the r and rb loci on starch grain size and on the content and composition of starch in developing pea seeds

The effects of mutant alleles at the r and rb loci on starchgrain size and the levels of starch and amylose in developingpea (Pisum sativum L.) seeds have been examined. Four lines,near-isogenic except for genes at these loci, have been usedto show that both mutations reduce levels of starch throughoutembryo development and reduce levels still further when combinedin the ‘double mutant’. The reduction in starchcontent was due, at least in part, to a reduction in starchgranule size. Although the proportion of starch in mature embryoswas similar in the rrRbRb and RRrbrb lines, the starch contentdiffered between these two lines during development, as a percentageof embryo dry weight. This difference was due to a reductionin the absolute growth of the embryo caused by the rb mutation.Lines homozygous for the mutant r allele with either wild-type(RbRb) or mutant (rbrb) alleles at the rb locus contained increasedproportions of amylose in their starch throughout development,due to a reduced production of amylopectin. The presence ofthe rb mutation, however, also reduced the amount of amylosein relation to the reduction in total starch levels. Mutantalleles at both loci also reduced starch levels in the testaduring development, the reduction due to rb being more extreme.Reciprocal crosses showed a maternal effect of the rb mutationon final seed size and on the absolute amount of starch in theembryo. Key words: Pisum sativum L., seed, starch, development, mutant     

Glycanases Associated with Cell Walls of Cicer arietinum L: Arabinogalactan Degradation

The proteins extracted from Cicer arietinum cell walls showedendo-glycanase activity against the water-soluble hemicellulosicpolysaccharides extracted with 4% KOH. Such endo-glycanase activitywas able to shift the molecular mass distribution of the arabinogalactancausing a decrease in its average molecular mass, as well asto release small oligosaccharides constituted exclusively ofarabinose. Exo-glycanase activities such as glucosidase andgalactosidase were also present. Key words: Arabinogalactan, cell wall, Cicer arietinum, glycanase, hemicellulose     

An Enzymic Basis for Pathogenic Specificity in Ophiobolus graminis

Avenacin, the glucosidic inhibitor present in oat roots, isacted on by a specific glucosidase produced by Ophiobolus-graminisvar. avenae, which destroys biological activity, as does hydrolysiswith 0.1 N. HCl. Neither O. graminis itself nor any other fungustested produces this enzyme. It is suggested that the resistanceof oats to O. graminis and its susceptibility to var. avenaedepend on an inhibitor-inactivating enzyme complex, and thisis compared with the antibiotic-inactivating enzyme complexfound in penicillin-producing moulds and resistant penicillinase-producingbacterial strains; such a complex may be concerned in othercases of pathogenic specificity. The way in which a varietyof O. graminis pathogenic to oats may have arisen is discussed.     

Mutant genes at the r and rb loci affect the structure and physico-chemical properties of pea seed starches

Mutant genes at two loci, r and rb, known to encode genes affectingthe starch biosynthetic pathway, were studied for their effecton the structure and gelatinization of pea seed starches. Comparisonswere made using starches from four lines {RRRbRb, rrRbRb, RRrbrb,and rrrbrb), near-isogenic except for genes at these two loci.All the starches had C-type X-ray diffraction patterns, butdifferent contents of ‘A’ and ‘B’ polymorphs.The presence of a mutation at either locus increased the ‘B’polymorph content in the starches, although the influence ofthe r mutation was much greater than that of rb. Differenceswere discovered in the crystalline stucture of the rrRbRb starchwhich correlated with a high content of amorphous phase as wellas with the changes in amylopectin structure. In addition, changesin the crystalline structure of this sample correlated witha lack of co-operative transition during starch gelatinizationin excess water. The RRrbrb starch had a greatly increased enthalpyof gelatinization in excess water compared with the wild-typestarch. It is proposed that this effect is connected with specificcharge interactions between the molecules in the starch granule.The rrrbrb starch had parameters of crystalline structure andgelatinization which reflected the different influences of thetwo genes. With regard to gelatinization, this starch had relativelywide co-operative transition and low enthalpy and a very highpeak temperature of transition. Key words: Pisum sativum, starch structure, genetic effects, rugosus mutants     

The Significance of Root Starch in Post-fire Shoot Recovery of the Resprouter Stirlingia latifolia R. Br. (Proteaceae)

Stirlingia latifolia, a common shrub of Banksia woodlands ofSW Australia, is a highly successful resprouter species recoveringfrom fire by multiple sprouting of new shoots from its upperroot stock. in comparison with the congeneric fire-sensitive(obligate seeder) species Stirlingia tenuifolia it exhibitsa low shoot:root dry weight ratio and high concentrations ofstored starch in the cortical tissue of its roots. The relationshipbetween root reserves of starch and development of newly sproutingshoot material following fire is examined in S. latifolia afterspring and summer burns. During the initial 2-5 month periodafter fire, levels of stored starch in the roots fall by 50-75%,followed by a slow increase as plants reproduce and the attainmentof pre-fire starch levels by 1·5-2 years after the fire.Starch reserves of roots can be further reduced by shading theregenerating shoots to limit their input of photosynthates andalmost totally eliminated by monthly removal of successive flushesof new shoots over a 10-12 month period. New shoots continueto sprout until all the starch is eliminated. The data are discussedin relation to the fire-induced reproduction of S. latifoliaand its ability to thrive in very frequently burnt habitats.Copyright1993, 1999 Academic Press Fire response, Proteaceae, resprouter, shoot:root ratio, starch storage, Stirlingia latifolia     

Seed Development in a Shrunken Endosperm Barley Mutant

Seg8 is one of eight barley (Hordeum vulgare L.) mutants whoseendosperm development is affected by the maternal plant genotype.This study was initiated to determine the nature and onset ofabnormal development to provide a basis for further studiesaimed at understanding the mechanism of genetic control. Seeddevelopment and synthesis and accumulation of reserve substanceswere compared between seg8 and its normal counterpart, cv. Klages.Light microscopic examination showed that the mutant phenotypeappeared as early as 4 d after anthesis (DAA), and seg8 graindry weight was significantly lower than cv. Klages by 8 DAA.Grain cell number was significantly lower in seg8 by 8 DAA,indicating an early termination of cell division. The mutanthad a lower starch concentration and higher sucrose concentration,also evident at 8 DAA. Rates of [¹⁴C]sucrose incorporation intostarch in excised half seeds were similar in both genotypesat 2 and 4 DAA, but at 8 and 12 DAA seg8 had a lower rate. Totalprotein concentration was not significantly different betweenthe two genotypes throughout endosperm development. These resultsindicate that the mutation affects cell division and starchaccumulation prior to 8 DAA. It is not known if the reductionin starch biosynthesis and accumulation results from a reducedcapacity for starch or a defect in starch biosynthesis. Hordeum vulgare L., barley, shrunken endosperm mutant, endosperm development, starch, protein, endosperm cell number     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Structure and Histochemistry of Stomata and Epidermal Cells in Five Species of Polypodiaceae

The epidermal structure of the five species of ferns, Arthromeriswallichiana (Spr.) Ching., Drymoglossum piloselloides (Prest.),Drynaria quercifolia (L.) J. Smith, Lepisorus nudus (Hook.)Ching. and Pyrrosia nuda (Gies.) Ching., has been investigated.Fifteen types of stomatal structures have been identified ofwhich copolo-desmocytic and coperi-desmocytic are new types.Four more possible stomatal structures: ccpolo-peri-, codesmo-polo-,codesmo-peri- and duplodesmocytic, are suggested. Localizationof starch, insoluble polysaccharides, protein and lipids hasbeen examined histochemically in the guard cells, subsidiarycells and epidermal cells. In Drynaria starch plastids and plastidscontaining both starch and protein are present in guard cells.Starch plastids are present in the subsidiary cells of all speciesexcept in Arthromeris, whereas, they are present in epidermalcells of only Drymoglossum and Lepisorus. Granular or amorphousinsoluble polysaccharides (other than starch) are present inguard cells of all the species, in the subsidiary cells of Arthromeris,Drynaria and Pyrrosia, and in the epidermal cells of Pyrrosia.Except in Pyrrosia lipids are present in the guard cells. Subsidiarycells of Drynaria and the epidermal cells of Arthromeris andDrynaria show lipid bodies. The presence of plasmodesmata andectodesmata is demonstrated in the epidermal cells of Drymoglossum.     

Characteristics of the Photosynthetic Metabolism of Carbon in Deuterated Chlorella ellipsoidea C-27

The photosynthetic metabolism of carbon in fully deuteratedcells of Chlorella ellipsoidea C-27 (D-Chlorella), obtainedby culture in medium prepared with 100 mol% D₂O, was characterizedby examining the activities of several enzymes and the levelsof metabolic regulators in a comparison with those of ordinarycells (H-Chlorella). The cellular content of starch in D-Chlorellawas more than twice that in H-Chlorella, whereas those of sucroseand glucose were significantly lower in D-Chlorella. Deuterationof Chlorella caused marked alterations in the activities ofenzymes involved in starch metabolism. There was a significantdecrease in the activity of phosphorylase, a catabolic enzyme,and a significant increase in the activity of starch synthase,an anabolic enzyme. These alterations are probably responsiblefor the increase in the amount of starch in cells. By contrast,no marked changes were observed in the activities of enzymesand the levels of metabolic inhibitors that are involved inthe synthesis of sucrose. It seems likely, therefore, that thedecrease in the amount of sucrose in D-Chlorella was causedmainly by a deficiency in sources of carbon in the cytoplasm,as a consequence of an increase in levels of starch in chloroplasts. (Received May 13, 1992; Accepted December 1, 1992)     

Correlation between the Starch Level and the Rate of Starch Synthesis during the Developmental Cycle of Chlorella ellipsoidea

The starch content as well as the rate of photosynthetic starchformation in Chlorella ellipsoidea was studied throughout thecell cycle. The starch level in Chlorella cells rose markedlyduring the growing phase in the light, but it started to decreaseafter about 14 to 16 hr regardless of illumination. The rateof starch synthesis, measured by the level of ¹⁴C-incorporationinto starch, increased rapidly in the growing phase until 10hr, and decreased promptly thereafter, even in the light. From these results, it was concluded that both the cellularlevel of starch and the rate of starch synthesis were a functionnot only of the light regime, but also of the stage of celldevelopment. ³ Present address: Yamada High School, Yamada-machi, Iwate Pref.028-13, Japan. (Received October 12, 1981; Accepted May 12, 1982)     

Expression of a-Amylase in Phaseolus vulgaris and Vigna mungo Plants

Levels of starch and soluble sugar in pods of Phaseolus vulgarisand Vigna mungo plants were analyzed during the course of maturationof fruits. The results suggest that the immature pods of P.vulgaris function to some extent as temporary reservoirs ofcarbohydrates for growth of seeds. A less clear pattern of accumulationof starch was observed in pods of maturing fruits of Vigna mungo.Measurements of a-amylase activites in pods of maturing fruitsand immunoblotting with an antiserum against     

Cellulolytic Activities of Botryodiplodia theobromae Pat

Botryodiplodia theobromae Pat., a ‘stain’ fungusisolated from stained and decaying wood of Bombax buonopozenseP. Beauv., attacked isolated blocks of Bombax wood. In theseit perforated the cell walls and formed elongated cavities withpointed ends, usually in chains, in the secondary wall. Thefungus attacked cellulose in a similar manner to soft rot fungi.It used starch and other saccharides present in the wood ofBombax buonopozense as initial substrates before degrading thecellulose and hemicellulose components of the wood. It did notdegrade the lignin component of the wood. Cellulase, ß-glucosidase, and amylase activities weredetected in culture filtrates of Botryodiplodia theobromae.Cellulase synthesis was delayed when the wood powder in themedium contained starch and saccharides. Amylase, too, behavedlike an inducible enzyme and disappeared from the cultures onexhaustion of starch. Electron microscopy showed that cellulose extracted from thewood of Bombax buonopozense was fragmented into small particleson treatment with the culture filtrates of Botryodiplodia theobromae.Cellobiose, glucose, and another saccharide, probably cellotriose,were identified as the products of enzymatic hydrolysis of cellulose. These results are discussed in relation to the attack and degradationof the wood of Bombax buonopozense by Botryodiplodia theobromaein nature.     

A mutation at the rb gene, lowering ADPGPPase activity, affects storage product metabolism of pea seed coats

Seed coat development was studied on two nearisogenic linesof peas (Pisum sativum L.): RbRb (wild type, round seed) andrbrb (wrinkled seed). A mutation at the rb locus modifies thedry seed shape and reduces the starch content of the embryo.This mutation is now known to affect the activity of ADPGlucosepyrophosphorylase, a key enzyme in the starch biosynthetic pathway.We have investigated the effects of the rb mutation on seedcoat development and found that the mutation reduces the growthrate and starch content in this organ. However, experimentson the kinetics of ¹⁴C-sucrose loading showed that starch synthesisfrom unloaded sucrose occurred in the seed coat for both mutantand wild-type lines. In addition, the sucrose concentrationwas increased and amino acid concentration decreased such thatthe nutritional balance of the embryos was affected. However,osmolality of the seed coat cells was not affected, suggestinga regulatory process which allows the maintenance of the importof assimilates in the seeds of either line. Key words: ADPGlucose pyrophosphorylase, seed coat, seed development, starch metabolism, wrinkled seed     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Effects of Light and Dark on the Ultrastructure of Lichen Algae

Quantitative ultrastructural observations have been made onthe algal cells (Trebouxia) in two lichens, Parmelia sulcataand P. laevigata, stored for 48 h in the dark or under a light/darkregime. The response of the alga was found to differ in theselichens. In P. sulcata the dark treatment caused a decreasein starch grains, lipid-rich pyrenoglobuli and peripheral cytoplasmicstorage bodies and an increase in pyrenoid and chloroplast proteinbodies. The algae in P. laevigata contained little starch andno chloroplast protein bodies. However, after dark treatment,starch, cytoplasmic storage bodies and pyrenoid dimensions sometimesdeclined, while pyrenoglobuli numbers increased. Some of theseapparent changes depended upon the units used for calculatingthe cross-sectional areas of structures, e.g. absolute units,percentage of cell wall, protoplast or chloroplast cross-sectionalarea. Chloroplast area increased in the dark (as a % of cellwall area) in both species while mitochondria were larger inthe dark in P. sulcata but not in P. laevigata. Ultrastructuralchanges were not clearly correlated with changes in photosyntheticand respiratory rates. These results directly support the suggestionthat some intra-cellular structures are energy-generating reservesthe dimensions of which can rapidly change. Parmelia sulcata, Parmelia laevigata, lichen algae, light and dark storage, starvation, reserve substances, organdie dimensions     

Starch Grain Casts and Moulds in Eocene (Tertiary) Fossil Mangrove Hypocotyls

Further examples of the recently reported fossil starch graincasts and moulds in the fossil mangrove Ceriops cantiensis Chandlerfrom the London Clay (Eocene) of the Isle-of-Sheppey, Kent,England have now been discovered. In addition, similar structureshave been found in the fossil hypocotyls of Palaeobruguieraelongata Chandler from Herne Bay, Kent and also in four recentlycollected hypocotyls of a new species of fossil mangrove fromSheppey. Starch grains from herbarium material of the extantmangrove genera, Bruguiera and Ceriops, have been examined forcomparison with the fossil forms. starch grain casts, fossil, mangrove, hypocotyls, palaeobotany, Eocene, framboids, Rhizophoraceae, Bruguiera sexangula, Ceriops cantiensis, Palaeobruguiera elongata     

Carbon Supply From Starch Reserves to Spring Growth: Modelling Spatial Patterns in Kiwifruit Canes

The remobilization and transport of reserve carbon from a kiwifruit(Actinidia deliciosa)cane to developing axillary shoots wasmodelled using a simple pool structure and mass flow process.Manipulative experiments with mature kiwifruit plants are comparedwith simulation results. The model uses detailed architecturalinformation rather than explicit partitioning functions, achievingcarbon partitioning as emergent behaviour of a spatial organization.The model successfully simulated shoot growth and starch distributionpatterns.Copyright 1999 Annals of Botany Company Carbon reserves, starch reserves, plant architecture,Actinidia deliciosa,kiwifruit.     

How Anti-Juvenile Hormones Work

SYNOPSIS. TWO compounds which possess anti-juvenile hormoneactivity in insects have been isolated and identified from theplant Ageratum houstonianum. These compounds, called precocenes,have been shown to inhibit the development of the insect corpusallatum (CA) and/or destroy the CA in sensitive species. Theaction of the precocenes can be seen to affect only those CAwhich are actively synthesizing and secreting the juvenile hormone.Structure-optimization studies of the precocenes reveal thatthe 3,4-double bond must be present and unsubstituted for biologicalactivity to occur. In vivo and in vitro studies reveal thatthe precocenes are very probably converted into reactive epoxideswhich undergo facile reaction with nucleophiles since histologicalexamination of precocene-inactivated CAs indicate a direct cytotoxicaction of the precocenes on the parenchymal cells of the CA.The most likely mode of action would appear to involve the alkylationof macromolecular cellular elements by a reactive epoxide orsimilar activated precocene intermediate.