Studies on the {beta}-glucosidase System of Trifolium repens L.

Using DEAE-cellulose two distinct ß-glucosidase enzymeshave been identified in white clover; one form is primarilyassociated with the seed, the other with young leaves. Evidenceis presented for the separate nature of ß-glucosidaseand ß-galactosidase activity in dry seed flour andin young leaf tissue. These results are considered in relationto the function of the Li locus in white clover, which is shownto control both ß-glucosidase and ß-galactosidaseactivity in expanding leaves.     

In-situ localization of cyanogenic β-glucosidase (linamarase) gene expression in leaves of cassava (Manihot esculenta Cranz) using non-isotopic riboprobes

The release of hydrogen cyanide (cyanogenesis) from damaged plant tissue depends upon the sequential action of a β-glucosidase and an α-hydroxynitrilase on cyanoglucosides. The non-isotopic digoxigenin labelling system was used to visualize the presence of cyanogenic β-glucosidase (linamarase) mRNA in cells of young leaves of Manihot esculenta Cranz (cassava). Strong hybridization to antisense riboprobes produced from the cDNA clone pCAS5, indicates localization of linamarase gene expression in laticifers (latex vessels). This is supported by the demonstration of linamarase mRNA in exuded latex. In contrast, in-situ localization of the control gene pGLF4, showed expression in all leaf mesophyll cells. High levels of linamarase activity were demonstrated in the latex of leaf petioles and this activity was shown to be dependent on the presence of attached leaflets. Assays of α-hydroxynitrilase activity in exuded latex and whole leaves shows that, unlike linamarase, this enzyme is present at very low levels in latex and must be located elsewhere in the leaf.     

Some Morphological and Chemical Characteristics of Developing Fruits of Raphia hookeri

Ndon, B. A. 1985. Some morphological and chemical characteristicsof developing fruits of Raphia hookeri.—J. exp. Bot. 36:1817–1830. Fruits which were at different stages of development were randomlysampled from different inflorescences of Raphia hookeri palms.The morphological characteristics and chemical (the dry matter,lipid and carbohydrate) contents of the exocarp and seeds weredetermined. The results showed that the seed length, circumferenceand volume were optimal at 24 months after pollination whichindicates that Raphia seeds attained maximum size at that period.The seed endosperm was liquid or semi-liquid between 6–18months after pollination but became solid with a prominent embryoat 24 months. The seed dry matter was low at the early stagesof development but there was a rapid increase in seed dry weightat 18–33 months after pollination. The seeds were physiologicallymatured at 30–33 months after pollination, while the exocarpmatured at 24–30 months after pollination. The Raphia seeds were low in lipid (about 2%) compared to theexocarp which had 30–40% lipid at full maturity. Maximumamount of lipid was accumulated within the exocarp at 36–42months after pollination and this period indicates the timefor harvesting Raphia fruits for maximum oil which is probablythe most economic part of the fruit. The total sugar concentration increased in the exocarp withincrease in maturity. Conversely the concentration of sugarsdecreased within the seeds as the fruit matured. Maximum totalsugar concentration (about 309 mg g^–1 dry fat free sample)was found in the exocarp at 36–42 months after pollination.Mature seeds at 48 months after pollination had about 50 mgof total sugars per g of fat free sample. There was insignificantaccumulation of starch in the exocarp. The mature seeds werelow in starch (5–10% of the dry weight). Key words: Raphia hookeri, development, fruit     

r{beta}-Glucosidase in the Indigo Plant: Intracellular Localization and Tissue Specific Expression in Leaves

rß-Glucosidase of indigo plant (Polygonum tinctorium)has a high substrate specificity for indican (indoxyl rß-D-gIu-coside).To examine the localization of this rß-glucosidase,we fractionated the cells of the leaves and analysed them im-munocytochemically.Immunoelectron micrographs with specific antibodies againstthe rßglucosidase clearly showed that the rß-glucosidasewas localized in the stroma of the chloroplasts in mesophyllcells, but not in the thylakoid membrane. Chloroplasts wereisolated from the crude ho-mogenate of the fresh leaves by Percolldensity gradient centrifugation and then subjected to suborganellarfrac-tionation. rßGlucosidase activity was specificallydetected in the stromal fraction, but not in the thylakoid membrane.This was also supported by the result of an immunoblot of thefractions with anti-rßglucosidase antibodies. Therß-gIu-cosidase was immunocytochemically localizedin the chloroplasts of mesophyll cells, but not in any chloroplastsin marginal cells of the vascular bundle or epidermal cells;ribulose 1,5-bisphosphate carboxylase (Rubisco), a typical stromalprotein, was observed in all chloroplasts in these cells. Theseresults suggest that rß-glucosidase is tissue specificin its expression in the leaves of the indigo plant. (Received April 14, 1997; Accepted July 10, 1997)     

Growth Rate and Water Relations of Citrus Leaf Flushes

Growth rates of seasonal leaf flushes of ‘Valencia’orange [Citrus sinensis (L.) Osbeck] were measured and waterrelations characteristics of young (new) and over-wintered (old)citrus leaves were compared. New flush leaves had lower specificleaf weights and lower midday leaf water potentials than comparablyexposed old leaves. Spring and summer flush new leaves had higherosmotic potentials than old leaves. These differences becamenon-significant as the new leaves matured. During summer conditions,water-stressed new leaves reached zero turgor and stomatal conductancealso began to decrease in them at higher leaf water potentialsthan in old leaves. Old leaves were capable of maintaining openstomata at lower leaf water potentials. Opened flowers and newflush leaves lost more water, on a dry weight basis, than flowerbuds, fruit or mature leaves. The results illustrate differencesin leaf water potential and stomatal conductance which can beattributed to the maintenance of leaf turgor by decreases inleaf osmotic potentials as leaves mature. These changes in citrusleaf water relations are especially important since water stressresulting from high water loss rates of new tissues could reduceflowering and fruit set. Citrus sinensis (L.) Osbeck, orange, Citrus paradisi Macf., grapefruit, growth rate, leaf water relations, osmotic potential, water potential, stomatal conductance     

Identification, Purification and Properties of the Precursor of Conglutin {beta}, The 7S Storage Globulin of Lupinus albus L. Seeds

Lupinus albus L. developing cotyledons 35 d after floweringcontained a major polypeptide of-average M_r 64000, immunologicallyrelated to conglutin ß, the 7S storage globulin ofthis seed. This polypeptide decreased during seed maturation,without completely disappearing in the mature seed. This dropwas accompanied by the formation of polypeptide fragments typicalof the mature conglutin ß. The 64000 polypeptide hasbeen identified as the precursor polypeptide of conglutin ß. Undenatured conglutin ß precursor, purified by ionexchange chromatography and size exclusion chromatography, showedsurface and association properties identical to the mature conglutinß molecule. The precursor oligomer, of M_r 190000,consisted of an association of three 64000 subunits. They stronglyreacted with concanavalin A indicating the presence of covalentlylinked carbohydrate. Tryptic treatment of the undenatured conglutin ß precursorled to the accumulation of a relatively stable 59000 polypeptidewhich was cleaved later on and produced three large polypeptidefragments differing from the mature conglutin ß polypeptides. Key words: Conglutin ß, precursor, developing seeds, Lupinus albus L.     

Acid phosphatase in plant tissues I. Changes in activity and multiple forms in tea leaves and tomato fruit during maturation and senescence

Multiple forms of acid phosphatase varied in tea leaves (Camelliasinensis L.) depending on variety and the age of the leaves.Polyacrylamide gel electrophoresis indicated the presence ofsix multiple forms in extracts of young leaves and seven inextracts of mature leaves of the variety Hatzumomizi. VarietyBenihomare showed five bands in zymograms of young-leaf extractsand six in zymograms of mature-leaf extracts. In addition tothe appearance of the new band, the relative intensity of stainingin two other bands changed during maturation. One decreasedand one increased in intensity. The components were partiallyseparated by CM-cellulose chromatography and isoelectric focusing.Total activity of acid phosphatase did not change greatly ona fresh-weight basis during maturation and aging. Extracts of immature green tomato (Lycopersicon esculentum Mill.)fruit showed six bands on polyacrylamide gels. Isoelectric focusingand subsequent electrophoresis of the fractions obtained atdifferent pH values, produced evidence for seven acid phosphatasecomponents in tomato extracts. Unlike the situation with certainother climacteric fruit, acid phosphatase in tomato extractsdecreased steadily during ripening to a level of activity only25–40% that of immature green fruit extracts. It is thereforeconcluded that the onset of senescence in climacteric fruitsis not dependent on an increase in acid phosphatase. Substratespecificity, response to inhibitors, Km, and pH optima of partiallypurified fractions were similar to those of tea-leaf extracts. (Received November 29, 1972; )     

Production of Cellulolytic Enzymes by Botryodiplodia theobromae, Pat.

Carboxymethylcellulase (CM-cellulase) and ß-glucosidaseactivities were induced in cultures of Botryodiplodia theobromae,Pat. Both growth of the fungus and CM-cellulase production werebetter with sodium nitrate as nitrogen source than with eitherammonium nitrate or ammonium chloride. Growth, per unit nitrogensupplied, was greater with glutamic acid and aspartic acid asnitrogen sources than with sodium nitrate; this was probablybecause the fungus utilized the carbon of these amino-acids.Most ß-glucosidase activity was formed with ammoniumchloride or ammonium nitrate as nitrogen source. Glucose inhibited the activity of ß-glucosidase markedly.Low glucose concentration (c. 0.003 M) stimulated CM-cellulaseactivity but concentrations above 0.05 M inhibited. The formationof both enzymic activities was repressed in the presence ofglucose. ß-glucosidase activity was more thermolabilethan that of CM-cellulase.     

Diet and nutrition in wild mongoose lemurs (Eulemur mongoz) and their implications for the evolution of female dominance and small group size in lemurs

Data collected on the feeding behavior, food intake, and chemical analyses of plant foods were used to document seasonal variation in diet and nutrition in Eulemur mongoz in northwestern Madagascar. E. mongoz conforms to the general Eulemur dietary pattern, with a predominantly frugivorous diet supplemented mainly by leaves, flowers, and nectar. Phytochemical analysis revealed high water contents in all the main plant foods; mature fruit and flowers contained the most water-soluble carbohydrates; immature leaves were richest in protein and essential amino acids; the limiting amino acids in all plant foods were methionine and cystine; ash (mineral) content was highest in petioles and mature leaves; crude lipid content was highest in seeds; and crude fiber content was indistinguishable between immature and mature fruit and leaves. High-fiber foods were eaten during both seasons; the wet season diet was dominated by high-energy foods (mature fruit, nectar, and seeds), while the dry season diet contained foods high in energy (mature fruit and flowers) and high in protein (immature leaves) and minerals (mature leaves and petioles). However, nutrient intake did not vary between seasons, implying that nutrient requirements are met throughout the year. These results suggest we draw more conservative conclusions when interpreting dietary variability in the absence of chemical analysis, and also draw into question the idea that nutritional stress is a factor in the timing of reproduction in lemurs and, by extension, is linked to the prevalence of female dominance and small group size in lemurs.     

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     

Identification of Jasmonic Acid in Three Species of Higher Plants and Its Biological Activities

(–)-Jasmonic acid (JA) was isolated and characterizedfrom immature seeds of Phaseolus vulgaris L. as a growth inhibitoragainst rice seedlings. The presence of JA was also confirmedin both fresh leaves and insect galls of Castanea crenata Sieb.et Zucc. and immature seeds of Dolichos lablab L. by combinedgas chromatography-mass spectrometry analyses. The biologicalactivities of JA were evaluated in several bioassay systems,and it was shown that JA is a new type of plant growth regulator. (Received February 9, 1981; Accepted March 27, 1981)     

Reconstitution of cyanogenesis in barley (Hordeum vulgare L.) and its implications for resistance against the barley powdery mildew fungus

Barley (Hordeum vulgare L.) produces a leucine-derived cyanogenic β-d-glucoside, epiheterodendrin that accumulates specifically in leaf epidermis. Barley leaves are not cyanogenic, i.e. they do not possess the ability to release hydrogen cyanide, because they lack a cyanide releasing β-d-glucosidase. Cyanogenesis was reconstituted in barley leaf epidermal cells through single cell expression of a cDNA encoding dhurrinase-2, a cyanogenic β-d-glucosidase from sorghum. This resulted in a 35–60% reduction in colonization rate by an obligate parasite Blumeria graminis f. sp. hordei, the causal agent of barley powdery mildew. A database search for barley homologues of dhurrinase-2 identified a (1,4)-β-d-glucan exohydrolase isozyme βII that is located in the starchy endosperm of barley grain. The purified barley (1,4)-β-d-glucan exohydrolase isozyme βII was found to hydrolyze the cyanogenic β-d-glucosides, epiheterodendrin and dhurrin. Molecular modelling of its active site based on the crystal structure of linamarase from white clover, demonstrated that the disposition of the catalytic active amino acid residues was structurally conserved. Epiheterodendrin stimulated appressoria and appressorial hook formation of B. graminis in vitro, suggesting that loss of cyanogenesis in barley leaves has enabled the fungus to utilize the presence of epiheterodendrin to facilitate host recognition and to establish infection.     

Pollination and Resource Constraints on Fruit Set and Fruit Size ofPersoonia rigida(Proteaceae)

Flower and immature fruit abscission can be caused by proximatefactors preventing the development of all flowers into maturefruits. Two potential limiting factors, pollen supply and carbohydrateavailability, were assessed as relative constraints on accumulationof fruit dry matter inPersoonia rigida(Proteaceae). Naturalpollen transfer was highly efficient, and no evidence of self-incompatibilitywas found. Levels of fruit set (67.4% following open-pollinationand 50.4–58.8% following hand-pollinations) may be thehighest recorded for a species of Proteaceae with hermaphroditicflowers. Fruit abscission occurred principally in a single phasefrom 4–10 weeks post-anthesis, with a peak during theeighth week post-anthesis, and this early abscission was theresult of fertilization failure in some flowers. Manipulationof carbohydrate availability by girdling and/or defoliationof branches provided evidence that carbohydrate supply affectedfruit set only when supply was very low. Fruit abscission dueto very low carbohydrate availability on defoliated brancheswas evident during the early abscission phase but was more pronouncedduring the second half of the fruit development period (>17weeks post-anthesis), coinciding with the period of greatestdry matter demand by the fruits. Minimal fruit abscission occurredduring this later period on leaf-bearing branches. Increasesin carbohydrate supply had no effect on fruit set, but fruitsize was highly sensitive to carbohydrate availability. Subtendingand adjacent leaves were identified as major contributors tothe carbohydrate supply of fruits, although some carbohydratecould be obtained from beyond the fruiting branch. Despite thehigh levels of natural fruit set, both pollen supply and resourceavailability were indicated as potential constraints on accumulationof fruit dry matter inP. rigida.Copyright 1999 Annals of BotanyCompany. Persoonia rigidaR.Br., Proteaceae, pollination, carbohydrates, fruit set, fruit size, abscission, girdling.     

The Gamete Pigments of Hormosira banksii (Phaeophyta)

The gamete pigments of Hormosira banksii have been separatedby thin-layer chromatography and identified by absorption spectrophotometry.Male gametes contain predominantly ß-carotene, andfemale gametes predominantly chlorophyll and fucoxanthin. Severalminor pigments have also been tentatively identified.     

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     

Scutellar Amylase Activity in Naturally Aged and Accelerated Aged Wheat Seeds

Scutellum extracts of dry unaged and differentially aged wheatseeds showed appreciable and ß amylase activities,these being higher in unaged and lower in aged seeds. This indicatesthat scutellar amylase may remain long-lived in seeds. Duringgermination of both unaged and aged seeds an increase in scutellaramylase (particularly ß) occurs. Initiation of thisgermination associated increase in amylase activity, however,was found to occur relatively late during imbibition. Further,while scutellar amylase increased considerably during imbibitionin unaged seeds, in differentially aged seeds the increase wassmall. This also contributes to the reduced scutellar amylaseactivities in aged seeds. Triticum aestivum L., wheat, naturally aged, accelerated aged, unaged, germination, scutellar and ß amylase     

Curd Initiation in the Cauliflower: I. JUVENILITY

Hand, D. J. and Atherton, J. G. 1987. Curd initiation in thecauliflower. I. Juvenility.—J. exp. Bot. 38: 2050–2058. Four cauliflower (Brassica oleracea var. botrytis L.) cultivarswere screened for differences in juvenility, measured as thephase of insensitivity to vernalization. Juvenility persisteduntil the initiation of a critical number of leaves which sharplydefined transition to the sensitive, mature form at 13 to 15leaves in cv. Perfection and at 17 to 19 leaves in cv. WhiteFox. Preliminary investigation showed transition in cv. AlphaCliro to occur between 9 and 18 leaves initiated and in cv.Dole after 19 leaves. Leaf number was a stable marker of theend of juvenility in plants grown under different light conditions,whereas time, leaf area and leaf dry weight were not. Leaf numberwas linearly related to log whole shoot dry weight. The rateof leaf initiation in plants of cv. Perfection growing duringthe juvenile phase was approximately one third that of plantsin the mature phase, when measured on a thermal time base. Chilling mature, vegetative plants at 5 °C for 28 d advancedcurd initiation by up to 35 leaves in cv. Perfection; 27 leavesin cv. White Fox; 27 leaves in cv. Alpha Cliro and 21 leavesin cv. Dok, compared with plants grown continuously at 20 °C. Key words: Cauliflower, vernalization, juvenility     

Studies on the Germination of the Seeds of Striga hermonthica: III. On the Nature of Pretreatment and after-ripening

An attempt has been made to elucidate some of the changes whichtake place in seeds of Striga hermonthica (immature, ripe andoverripe), (a) during moisture-treatment at three temperatures(15°C., 22°C., and 32°C.), and (b) during after-ripeningin dry storage. Observations on the drift of respiration of immature seed throughoutpretreatment at 15°C. show that an inverse relationshipexists between germination and respirational activity. Thissuggests that germination depends on the accumulation of somemetabolite during pretreatment. It would seem that this metabolitemay also be used as a substrate for respiration during pretreatment. After-ripening of the immature seeds has been shown to changethe nature of the seeds in some way so that their peak respirationon exposure to moisture is at first progressively reduced. Thisreduction has been ascribed to the presence of some barriercapable of restricting gaseous diffusion. The data presented, togetherwith those of the previous paperof this series, suggest that the specific metabolite requiredfor germination might be proteinaceous. Certain other data appearto rule out the suggestion that germination capacity is conditionedby the sugar content of the seeds. Reasons are given which supportthe view that one of the main effects of the stimulating solutionon the seed is to increase the permeability of the seeds togaseous diffusion.     

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.