Distribution of Glycosidases and Acid Invertase Activities in Relation to Elongation Growth in Pearl Millet Internode

The mean cell length along a differentiating internode and alliedchanges in the activities of ß-glucosidase, - andß-galactosidase. -mannosidase and acid invertase,together with the contents of reducing and non-reducing sugars,were examined in pearl millet (Pennisetum americanum L. Leekecv. BJ-104). The specific activities of cytoplasmic -mannosidase,wall ß-glucosidase, and cytoplasmic and wall acidinvertase showed close relationships with the rate of cell elongation.The linear regressions of the rate of cell elongation, and thespecific activities of wall ß-glucosidase and cytoplasmicand wall invertase showed significant positive correlations(P<0·05), whereas cytoplasmic -mannosidase was negativelycorrelated (P<0·01). The results are discussed in the light of cell wall looseningand the provision of carbon substrates for cell elongation. Key words: Glycosidases, acid invertase, sugars, cell elongation, Pennisetum americanum L., Leeke     

Starch Deposition and Carbohydrase Activities in Developing and Germinating Soya Bean Seeds

Immature soya bean seeds accumulate starch as a transient reservematerial which is utilized later in development. Germinatingseeds also accumulate starch reserves, probably as a resultof gluconeogenesis from storage lipid. Developing beans showa rapid increase in ß-amylase activity which continuesinto early germination before declining. Distribution of ß-amylaseactivity is not consistent with its supposed role in starchdegradation. Soya bean seeds also contain -amylase and -glucosidaseactivities which could be responsible for starch mobilization. Glycine max (L.) Merr., soya bean, starch, carbohydrase, amylase, -glucosidase     

Glycoprotein Nature of Glycosidases from Leaves of Pisum sativum L

-Mannosidase, ß-N-acetylglucosaminidase, - and ß-galactosidaseand ß-glucosidase were partially purified from leavesof Pisum sativum by ammonium sulphate fractionation and columnchromatography on DEAE-Sephadex A-50 and hydroxylapatite. Atleast two molecular forms of each enzyme were resolved by thesetechniques except for ß-glucosidase of which onlyone form was resolved. Except for one form of -galactosidase,all of the glycosidases thus purified were completely boundby Sepharose-linked Concanavalin A. The binding was stronglyinhibited by cr-methyl-D-mannoside and no binding to Sepharose-6-Boccurred indicating that these glycosidases contain mannose-richoligosaccharides. The glycoprotein nature of -mannosidase, ß-galactosidaseand ß-glucosidase was further demonstrated by chromatographyon phenylboronate agarose columns. The differences in the concentrationof cr-methyl-D-mannoside and sorbitol required to elute thevarious glycosidases from Sepharose-linked Con A and phenylboronateagarose, respectively, suggested that these enzymes are glycosylatedto various degrees or that structural variation in their carbohydratemoieties occur. This is the first demonstration that glycosylationof several glycosidases present in a single plant species isapparently a generalized feature of these enzymes. Key words: Pisum sativum, Glycosidase, Glycoprotein     

Occurrence of Multiple Forms of {alpha}-Amylase and Absence of Starch Phosphorylase in Soya Bean Seeds

Soya bean cultivars ‘Altona’ and ‘Chestnut’have active but quite low levels of -amylase. Activity was assayedwith specific substrates, oxidized amylose and ß-limitdextrin, which were resistant to attack by ß-amylase.During seed development -amylase activity increased to a maximumin both cultivars and then declined towards maturity. Matureand germinating seeds retain low activities of -amylase. Gelelectrophoresis separated the -amylase activity into six majorbands which occurred in both cultivars. The isozyme patternwas quite similar for developing, mature and germinating seed.although the relative proportion of activity in the variousbands changed somewhat. Starch phosphorylase was not detectedin any soya bean seed samples tested, but good activity wasfound in potato tuber extracts used as a control. Mixing experimentsusing soya bean and potato extracts indicated there were noinhibiting factors in soya bean seed extracts. Soya bean seedextracts probably do not contain starch phosphorylase. Glycine max (L.), Merr, soya bean, -amylase, isozymes, phosphorylase     

Glycosidases in Carrot Cells in Suspension Culture: Localization and Activity Change during Growth

Localization of four glycosidases, -galactosidase (-Gal), ß-galactosidase(ß-Gal), -glucosidase (-Glu) and ß-glucosidase(ß-Glu) in suspension-cultured carrot cells was studied.Wall-bound enzymes were made soluble when the cells were convertedto protoplasts by cellulase and pectinase. -Gal was separatedinto two forms, designated I and II, by chromatography on aSephadex G-200 colunm. -Gal I was located exclusively in thecytoplasm whereas -Gal II was found in both the cytoplasmicand cellwall fractions. The pH optimum was in the neutral regionfor -Gal I and in the acidic region for the other glycosidases,including -Gal II. Both intact cells and protoplasts in suspensionculture secreted these glycosidases, except -Gal I, into themedium. Specific activities of the glycosidases, especiallythe activity of ß-Gal, decreased in the early logarithmicgrowth phase and increased as cells went through late logarithmicand stationary phases. In protoplast culture, glycosidase activitygradually increased as cell wall regeneration proceeded. (Received December 13, 1980; Accepted February 10, 1981)     

Changes in Carotenoid Composition of Certain Roses with Age

The carotenoid composition of the anthers and styles and thefruits of four roses of different ages have been investigated.ß-carotene and its epoxides were present at all stagesstudied, as were the ß-carotene derived xanthophyllauroxanthin and the -carotene derived flavoxanthin and chrysanthemaxanthin.As the various parts aged, the control of carotenoid synthesiswas removed, oxidative processes took place with the resultthat very few members of the -carotene series were found, andepoxy-carotenoids and their derivatives were the main carotenoidspresent. Rubixanthin, 3-hydroxy--carotene characteristic ofrose hips was found in fairly large amounts in anthers and stylesand -carotene was probably the precursor of this pigment. Rosehips were also investigated for their vitamin-A contents whichwere not as high as those of All Gold flowers.     

Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

Carotenoid Pigment Changes in Ripening Momordica charantia Fruits

The carotenoid composition of Momordica charantia fruit (pericarp)at four levels of maturity was extensively investigated. Thenumber of carotenoids isolated increased from five in the immaturefruit to six at the mature-green and 14 at the partly-ripe andripe stages. Cryptoxanthin, which could not be isolated at theimmature and mature stages, accumulated rapidly at the onsetof ripening to become the principal pigment of the ripe fruit.Moderate increases were seen in ß-carotene, zeaxanthinand lycopene concentrations as ripening progressed. The reversetrend was observed with lutein and -carotene which were themajor pigments of the immature fruit. Prior to the colour break,only the hydroxy derivatives of -carotene (zeinoxanthin andlutein) could be detected; the ß-hydroxy compounds(cryptoxanthin and zeaxanthin) appeared and predominated thereafter.The hydroxy carotenoids of the ripe fruit were almost entirelyesterified in contrast to those of the unripe fruit which weremainly unesterified. Traces of flavochrome, 5, 6-monoepoxy-ß-carotene,mutatochrome, -carotene, -carotene, -carotene and rubixanthinwere detected in the partly-ripe and ripe fruits but not inthe immature and mature-green samples. Phytofluene was observedin trace levels at all stages.     

Dry Matter Distribution between the Shoot and Storage Root of Carrot (Daucus carota L.): I. Comparison of Varieties

The distribution of dry matter between shoot and storage rootof a wild variety and cultivars of carrot was investigated ina glasshouse experiment and a field experiment. In both experimentsthe deliberate introduction of variation in plant size revealeda linear relationship between the log weights of these partsat any time throughout growth. In the glasshouse experiment,in which growth was examined from approximately 10–600mg mean dry wt per plant, the slope of the relationship betweenIn shoot and In storage root dry wts decreased markedly duringdevelopment to 100 mg. Subsequently the slope became reasonablyconstant and the intercept progressively more negative. Datafrom the field experiment covered a similar timescale (48–115days) to the glasshouse experiment but the plants grew morequickly and were much heavier (180 mg–6 g dry wt). Therelationship between shoot and storage root for these data wassimilar to that for the plants of comparable size in the glasshouseexperiment. Over the range of development for which the slopeof the relationship was constant, data from both experimentswere described fairly well by the following equations: In shoot = – time+ In storage root where , and are constants. Between cultivars the slope ()and time constant () were similar, but the intercept () increasedwith maturity time of the cultivars. For wild carrot the slopewas larger and the time constant smaller than estimates forthe cultivars. These results on dry matter distribution arediscussed in relation to a model for partitioning assimilatein an individual plant. Daucus carota L., carrot, assimilate distribution, dry matter distribution, storage root, partitioning model     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

{gamma}-Aminobutyric Acid Metabolism in Plants: Part 2. Metabolism in Higher Plants

The metabolism of -aminobutyric acid (AB) has been studied inhigher plants, particularly in peas and peanuts. Transaminationappeared to form the first step in AB degradation although transaminaseactivities were very low. The relatively active AB transaminaseassociated with whole pea plants possessing nodulated rootsappears to reside almost entirely within the nodules. AB transaminationwas demonstrated conclusively in extracts of mitochondria fromcotyledons of peanut seedlings; pyruvic acid acted as a betteramino-group acceptor than -ketoglutaric acid (KG). AB transaminaseactivity present in the microsomal and soluble cytoplasmic fractionsof the cells was very low AB was not metabolized perceptibly by intact mitochondria frompeanut, but when various organic acids were supplied simultaneously,an extra uptake of oxygen occurred and was associated with ABdisappearance. Aspartate, alanine, and ammonia were formed usingthe nitrogen atom of AB. The metabolic pathway followed by the carbon skeleton of ABwas traced by supplying C¹⁴-labelled material to leaf discsof peas and to mitochondria from peanut cotyledons. Radioactivitywas incorporated into organic acids, amino-acids, and respiratorycarbon dioxide in a manner suggesting that AB was convertedinto succinate which was then metabolized by the enzymes ofthe Krebs cycle present in the plant mitochondria. Glutamic decarboxylase was shown to be present largely in thenon-particulate (soluble) cytoplasm of cells. The enzymes responsiblefor AB synthesis and degradation, glutamic decarboxylase, andAB transaminase, respectively, therefore largely reside in differentsub-cellular fractions.     

Wilting of Leaves in Vicia faba L.

Wilting of leaves of Vicia faba L., which occurs when the pressurepotential (_p) is zero, and the leaf-water potential () at wiltingboth depend entirely upon the solute potential at incipientplasmolysis (_so) and not on soil-water status. Wilting in V.faba is acropetal; this is consistent with the hypothesis thatthere is a gradient of decreasing _so up the plant and that wateris transferred from the lower to the upper leaves, hasteningthe overall water loss from the lower leaves to the point when_p is zero. The gradient in _so up the plant is of the order of3–8 bar. It is proposed that wilting when _p>0 (i.e. > _so) shouldbe ‘apparent wilting’ and that when _p0 (i.e. _so),‘true wilting’.     

Pumpkin (Cucurbita sp.) seed globulin III. Comparison of subunit structures among seed globulins of various Cucurbita species and characterization of peptide components

Various Cucurbita seed globulins showed patterns similar toone another on SDS-gel electrophoresis, and ß bandsfor unreduced globulins and , ', and ' bands for reduced ones.On gel electrophoresis in 6 M urea, reduced globulin gave twoacidic and two basic bands. These corresponded to and ' chainsand ₁ and ₂ chains, respectively, identified by two-dimensionalurea-SDS gel electrophoresis. The compositions of the and ßsubunits were proposed. (Received September 8, 1977; )     

Pumpkin (Cucurbita sp.) seed globulin V. Proteolytic activities involved in globulin degradation in ungerminated seeds

Two proteolytic activities I and II involved in the globulindegradation were detected in pumpkin seeds. Activity I, hydrolyzing and ß subunits of the globulin to form F_ß,was found in both dry seeds and cycloheximide-treated cotyledons,and decreased during germination. Activity II, hydrolyzing F_ßto produce small peptides and amino acids, was not observedin dry seeds but found in cycloheximide-treated cotyledons,increased up to 4 days, and gradually decreased during germination. Activity I gave limited hydrolytic products from the globulinand the chain, but not from F_ß, the chain and some animal proteins. It was inhibitedby EDTA. On the other hand, activity II hydrolyzed F_ßand the chain faster than the globulin, the chain and some animal proteins. It was inhibitedby EDTA and p-chloromer-curibenzoate, and activated by ß-mercaptoethanol,dithiothreitol and CoCl₂. Optimum pH's were at about 6.8 andat 6.0 to 6.8 for activities I and II, respectively. The degradation process of the globulin can be divided intotwo steps: the first step is the conversion of globulin to F_ßand the second step, F_ß to small peptides and aminoacids. (Received November 9, 1979; )     

{gamma}-Amiobutyric Acid Metabolism in Plants: Part 1. Metabolism in Yeasts

The metabolism of -aminobutyric acid (AB) by two yeasts, Saccharomycescerevisiae and Torulopsis utilis, was investigated. Both yeastsgrew well upon AB as a sole source of nitrogen (N), and thelag phase for Torulopsis was shorter than when provided the N-source. The metabolism of AB by Torulopsis, whichwas associated with an increased O₂ uptake, was adaptive incharacter. The enzyme whose formation was induced by the supplyof AB was a transaminase, which was apparently specific forAB as the amino donor. Small amounts of transaminase were presentin unadapted, -grown cells. The optimum pH, equilibrium constant, Michaelis' constant, and coenzyme requirementwere investigated for the transamination reaction involving-ketoglutaric acid (KG) as amino group acceptor. Succinic semi-aldehyde(SSA) was a product of this transamination reaction.The possibility;that some AB was converted into SSA by a direct oxidative deaminationremained unconfirmed. The further conversion of SSA into succinic acid was establishedusing intact. cells for both yeasts. This oxidation processwas shown to be linked to the reduction of pyridine nucleotidesvising extracts of Saccharomyces as a source of SSA dehydrogenase.Dehydrogenase activity could be ascribed to two separate enzymes,one linked to DPN, and the other utilizing TPN and requiringMg⁺⁺ as an activator. The properties of the former enzyme, whichwas more important quantitatively, were investigated and comparedwith those described in the literature for an aldehyde dehydrogenaseof baker's yeast and for SSA dehydro-genases of Pseudomonas.Torulopsis extracts could catalyse the reduction of SSA to -hydroxybutyricacid (OHB); the OHB dehydrogenase involved required TPNH asa coenzyme. Certain other properties of this enzyme are recorded. The possibility is discussed that AB and SSA act as intermediatesin a metabolic pathway that may form a by-pass of the KG-succinatestage of the tricarboxylic acid cycle.     

Water-Relations Parameters of the Phloem. Determinations of Volumetric Elastic Modulus and Membrane Conductivity using an Applied Force Method and Shrinkage and Swelling of Tissues in Solutions at Differing Osmotic Pressure

Water-relations parameters were measured on sections of secondaryphloem from red oak (Quercus borealis michx. f.) and white ash(Fraxinus americana var. biltmoreana [Beadle] J. Wright) usinga linear displacement transducer. Changes in tissue thicknessin response to changes in the osmotic pressure of the bathingsolution were used to calculate the volumetric elastic modulusplus osmotic pressure (_v + ) of the tissue, and an applied forcemethod was used to estimate the time constant for water equilibration(T). The hydraulic conductivity of the cell membranes (L_p) wascalculated utilizing _v + and r values. The time-dependent behaviour of the tissue was much more complexthan originally expected. A correction for a time-dependentprocess that we call ‘drift’ was required to obtainnumbers for _v + . Furthermore, _v + was calculated on two assumptionsin order to relate changes in tissue dimensions to sieve elementparameters. In the first case, a lower limit for _v + of thesieve elements was determined by attributing all changes intissue dimensions to these cells. For red oak the average _v+ on this assumption is 72 bars. Assuming that all cell typeswere equally responsible for the changes in tissue dimensionsresulted in an _v + value of 192 bars for oak. If _v + and rare the same for all cells in the tissue, L_p for the sieve elementsof oak is 9.6 x 10^–8 cm s^–1 bar^–1. Exudationfrom the sieve elements of white ash during excision of thephloem led to artificially high values of _v + for that species. Quercus borealis michx. f., Fraxinus americana var, biltmoreana (Beadle) J. Wright, red oak, white ash, water relations, phloem, volumetric elastic modulus, membrane hydraulic conductivity     

Genotypic Differences in Leaf Water Relations between Brassica juncea and B. napus

Various kinds of measurement of tissue water status were madeseveral times during water stress and recovery in Brassica juncea(cv Canadian Black) and B napus (cv Drakkar) Unstressed plantsof the two species had similar leaf water potentials (_w), solute(_s) and turgor potentials (_p) Values of relative water content(RWC) and the slope of the linear relationship between _p andRWC (_p/RWC) were greater in B napus than in B juncea Statistical correlations of pooled data for the watered andstressed treatments differentiated the relationships among RWC,_w and its components in the two species The major statisticaldifference was that _p/RWC was related to RWC in B napus andto _w and _s in B juncea A decline in _p/RWC with decreasing _sin B juncea may be a mechanism for maintaining _p at low soilwater potentials through maintenance of more elastic cell walls. Brassica juncea, Brassica napus, osmotic adjustment, tissue elasticity, water relations     

An Assessment of Gibberellin Structure-activity Relationships

Information on the biological activities of gibberellins (GAs)in the barley aleurone, Tangin-bozu dwarf rice, dwarf pea, lettucehypocotyl and cucumber hypocotyl bioassays is reviewed and discussedin the context of GA structure-activity relationships. The barley aleurone bioassay exhibits a limited response toGAs and it is suggested that this may be because the aleuronecells are able to carry out few GA interconversions. Consequentlyactivity is determined by the degree of compatibility betweenthe GAs and a receptor site. In this assay high biological activityis associated with GAs having a 3ß-hydroxy--lactonestructure. This activity is substantially enhanced by the additionalpresence of a 13-hydroxyl group. The substitution of a -lactoneor a -lactol for a -lactone results in reduced activity while3ß,13-dihydroxy GAs with either 20-carboxyl or 20-methylfunctions are completely inactive. The Tanginbozu dwarf ricebioassay responds to many more GAs than the barley aleuronesystem possibly because the rice seedlings can carry out extensiveGA interconversions. Under these circumstances GAs that areinactive per se can be metabolically converted to active forms.There is no interaction between the 3ß- and 13-hydroxyfunctions of GA molecules in the rice assay. Activity appearsto be determined by the degree oxidation of the C-20 group.The order of activity is usually -lactone > -lactol >-lactone > methyl > carboxyl. It is suggested this mayindicate that in rice seedlings C₂₀-GAs are converted to C₁₉-GAsvia a Baeyer-Villiger type oxidation. Activity in the dwarfpea bioassay is dependent upon GAs possessing both 3ß-and 13-hydroxyl groups and is again related to the state ofoxidation at the C-20 locus. In the lettuce bioassay activityis restricted to GAs with a -lactone function. In some instancesa -lactone, but not a -lactol, can substitute effectively. Thismay imply that the applied C₂₀-GAs are not converted to C₁₉-GAsand that the response to the -lactone results from the six-memberedring mimicking the -lactone at the receptor site. Only GAs havinga 19,10 or a 19,20 lactonic bridge show substantial activityin the cucumber bioassay. The additional presence of eithera 12- or a 13-hydroxyl group severely reduces activity.     

The Theory of Diffusion of Solutes in Pollen; Theory, Design and Experimental Procedure

The theory of diffusion of solutes in pollen is developed andused to derive formulae from which the mean diffusion coefficient() can be determined. A mathematical relation ß_PGC t = ln (C₀/C_f) is developedby employing Fick's law. In this relation is the average diffusion coefficient, ß_PGC is a constantcharacteristic of the pollen, the ‘Pollen Grain Constant’,C₀ and C_f are the initial and final concentration differencesbetween the solution of the solute and the pollen grain andt is the time of diffusion in seconds. A diffusion apparatushas been designed and made. The design is simple to operateand an experimental procedure is described to determine theß_PGC using dilute potassium chloride solutions. Knowingthe pollen grain constant, the method to determine diffusioncoefficient () with any other solute is explained. Diffusion, design, pollen, solutes, theory     

Effects of {alpha}-Hydroxysulphonate on Respiration of Mung Bean Root Tips

Hydroxysulphonate (-HPMS) was found to have an inhibitory effecton dark respiration of mung bean root tips. Inhibition was greatestin regions nearest the apex. The inhibition of root respirationby -HPMS was not due to an inhibition of glycolic acid oxidase.It is suggested that the action of -HPMS was probably on acetylCOA synthetase.