Pollen Development and Quantitative Cytochemistry of Exine and Intine Enzymes in Sunflower, Helianthus annuus L

High resolution cytochemical methods have been used to characterizepollen development and pollen-wall structure in Helianthus annuus.Aniline-blue fluorescent material, presumably callose, was detectedin the nexine layer throughout its development. It was associatedwith acid phosphatase activity, while the outer sexine possessedintense esterase activity during the young spore period. Acidphosphatase and esterase were present in both intine and exinewall sites in contrast to their specific location in other pollentypes. Quantitative cytochemical estimates of enzyme activityduring development reveal esterase patterns typical of gametophyticsynthesis, while acid phosphatase patterns are characteristicof sporophytic origin suggesting tapetal transfer during thevacuolate period. Helianthus annuus L, sunflower, pollen development, exine enzymes, intine enzymes, quantitative cytochemistry     

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase(PG) gene previously isolated from the flower bud of Chinesecabbage (Brassica campestris L. ssp. chinensis Makino, syn.B. rapa ssp. chinensis). This gene was found to be expressedspecifically in tapetum and pollen after the tetrad stage ofanther development. Antisense RNA technology was used to studythe function of BcMF2 in Chinese cabbage. Scanning and transmissionelectron microscopy revealed that there were deformities inthe transgenic mature pollen grains such as abnormal locationof germinal furrows. In addition, the homogeneous pectic exintinelayer facing the exterior seemed to be overdeveloped and predominantlyoccupied the intine, thus reversing the normal proportionaldistribution of the internal endintine layer and the externalexintine layer. Since it is a continuation of the intine layer,the pollen tube wall could not grow normally. This resultedin the formation of a balloon-like swelling structure in thepollen tube tip in nearly 80% of the transgenic pollen grains.Premature degradation of tapetum was also found in these transgenicplants, which displayed decreased expression of the BcMF2 gene.BcMF2 might therefore encode a new PG with an important rolein pollen wall development, possibly via regulation of pectin'sdynamic metabolism. Key words: Brassica campestris, Brassica rapa, Chinese cabbage, intine, PG, polygalacturonase, pollen wall Received 28 August 2008; Revised 14 October 2008 Accepted 20 October 2008     

The Apertural Wall in Pollen of Linum grandiflorum

The apertural wall in tricolpate pollen of Linum grandiflorumwas investigated in order to understand its functioning duringdesiccation and rchydration. Whole and sectioned pollen grainswere studied with light or electron microscopy and by cytochemicalmeans. The areas of the apertures were examined in fresh drypollen, in grains moistened on agar gel or removed from compatiblestigmas, and in pollen from mature undehisced anthers The intine was found to consist of an inner ß-glucanlayer and an outer pectic layer. At the apertures the pecticlayer is thickened and overlaid by a ß-glucan layer.The pectinaceous intine stains red with basic fuchsin. The presenceof a third wall layer, the medine, was not confirmed. The aperturalintine thickenings possess considerable imbibitional capacityand at rehydration they appear as swollen lenticular bodies A procedure is described for obtaining intact exine free grains(EFG's) and whole, separated exines of L. grandiflorum. Invariably,the released EFG's consisted of protoplasts encased in the cellulosicintine. In most grains the outer intine remained attached tothe separated exine In L. grandiflorum the outer wall of the aperture expands whilethe protoplast and endintine are still infolded. Apparently,the exintine becomes detached from the endintine during desiccationand re-attaches at rehydration. It is suggested that the transientdetachment controls the influx of water into the vegetativecell Except for morph-specific exine processes no differences instructure of the aperture wall or its functioning at rehydrationwere observed between pin and thrum grains Pollen wallM, apertures, exintine, exine free grains, rehydration, desiccation, Linum grandiflorum     

The Microfibrillar Component of the Pollen Intine Some Structural Features

The microfibrillar polysaccharide component of the pollen intinecan be isolated by progressive chemical digestion of the exineand the cellular contents and the extraction of the matrix materials.The resulting intine ‘ghosts’ reveal various characteristicstructural features. The microfibrils have apparent individualdiameters in the range of 5–15 nm, but they are commonlyassociated laterally to form ribbons, or aggregated in strandsor cables of dimensions great enough to be resolved with theoptical microscope. These often show preferred orientations,which can be associated with pollen grain shape and with thedisposition-of the germination apertures. The apertural intinemay be structurally complex, as in Abutilon hybridum, where,after the removal of the exine, the polysaccharide caps whichoverlie the protein storage sites of the pollen grain wall retainthe elaborate patterning of the original cytoplasmic evaginationsfrom the vegetative cell. Secale cereale, Narcissus pseudonarcissus, Abutilon hybridum, Crocus vernus, pollen grain, intine, exine, wall pattern, germination apertures, polysaccharide microfibrils, fluorescence microscopy     

Reproduction in Seagrasses: Nature of the Pollen and Receptive Surface of the Stigma in the Hydrocharitaceae

This paper describes the characteristics of the pollen and thereceptive surfaces of the stigmas in the three marine angiospermsincluded in the Hydrocharitaceae. The pollen in Enhalus acoroidesand Thalassia hemprichii is spherical and has an ornamentedexine. An exine layer is not found in Halophila stipulacea wherereniform pollen grains are contained within transparent moniliformtubes. Cytochemical tests show that the pollen wall in the threespecies contains acidic and neutral polysaccharides and acidhydrolase acitivity is detected in the intine of H. stipulaceaand T. hemprichii. In Thalassia, one of the intine enzymes,acid phosphatase, is unambiguously associated with cytoplasmicinclusions. Flowering in Thalassia is coincident with the spring tides andthe pollen is released as a mass suspended in a thecal slimewhich contains approximately 5 per cent by weight carbohydrate,the principal mono-saccharide being mannose. Electrophoreticanalysis of the pollen-free slime shows a single glycoproteincomponent. The stigmas of the three seagrasses are papillate and of the‘dry’ type possessing a continuous protein-aceouspellicle subtended by a cuticle. The stigma pellicle exhibitscytochemically detectable esterase activity and binds the lectinconcanavalin A. Acid phosphatase activity is localized beneaththe cuticle at the tips of the stigma papillae. The discoveries show that the characteristics of the pollenand stigmas in the seagrasses are comparable with those foundin terrestrial flowering plants. The similarity in enzymaticproperites of the pollen wall and stigma pellicle suggests that,intriguingly, a similar mechanism of cuticle erosion might wellfollow compatible pollination both on land and in the sea. Enhalus acoroides, Thalassia hemprichii, Halophila stipulacea, Halophila decipiens, seagrasses pollen wall, stigma surface, hydrolytic enzymes     

Germination of Monocolpate Angiosperm Pollen: Evolution of the Actin Cytoskeleton and Wall during Hydration, Activation and Tube Emergence

The monocolpate pollen grain of Narcissus pseudonarcissus L.has two preferred sites for tube emergence, one at each endof the colpus. While the cellulosic microfibnls of the innerlayer of the intine are disposed circumferentially in the centreof the grain, the microfibrils in these terminal sites are shorterand randomly oriented Soon after the beginning of hydration,inclusions of the vegetative cell begin movement, firstly ina rotatory manner, and then in a pattern focused on one or bothgermination sites, where the intine bulges as hydration progresses.These changes are associated with the evolution of the actincytoskeleton. Actin is present in the unactivated grain in theform of fusiform bodies. During hydration these dissociate toform finer fibrils, initially randomly disposed. Then, correlatedwith the change of the pattern of movement in the vegetativecell, the actin fibril system becomes polarized towards thegermination sites, where shorter fibrils accumulate. Callose,absent from the ungerminated grain, is deposited within thecellulosic wall in these locations, forming a shallow dome whicheventually develops into an annulus subtending the inner calloselining of the emerging tube. The transition to cylindrical growthis associated firstly with the development of zonation in thecytoplasm of the vegetative cell, with the tip occupied by apopulation of wall precursor bodies (P-particles) and a denseaggregate of short actin fibrils; and then with the establishmentof the ‘inverse fountain’ pattern of movement characteristicof the apical part of the extending tube. Narcissus pseudonarcissus L, pollen activation, pollen germination, actin cytoskeleton, tip-growth system, pollen-tube wall development     

Purification of C-Type Cytochromes from a Thermophilic Blue-Green Alga, Synechococcus vulcanus, by Hydrophobic Column Chromatography using Toyopearls

Two C-type cytochromes, c-550 and c-553, were extracted by animproved procedure from a thermophilic blue-green alga, Synechococcusvulcanus, and effectively purified by a two-step hydrophobicchromatography. The first step was performed with a ToyopearlHW-65C :ammonium sulfate-66 column and the second with a butyl-Toyopearl650 column. This work is the first to apply butyl-toyopearl650 to protein purification. (Received July 2, 1984; Accepted September 13, 1984)     

Pollen Wall Development in Gibasis (Commelinaceae)

The development of the one and-inline of the pollen wall aredescribed for Gibasis karwinsk yana and G. venustula. Duringthe tetrad stage the appearance of electron-opaque depositionsor tri-partite plates at discrete sites between the plasma membraneof the spore and the inward surface of the callose special wallare the first indications of exine development. The sulcus rapidlydifferentiates being composed of discrete exine granules ona thin foot layer. Probacula in non-apertural areas developin an electron-opaque granular layer situated between the plasmamembrane, which is highly convoluted, and the callose specialwall. A foot layer is formed from electron-opaque lamellae atthe plasma membrane. Exine pattern is clearly established withinthe tetrad. After release of the spores from the tetrad an intimate associationis rapidly developed between the plasma membrane of the periplasmodialtapetum and the newly-formed exine. Compacted electron-opaquematerial is found at the interface between membrane and theexine and vesicular material is added from the tapetum. Theincrease in volume that occurs in both spore and anther is accompaniedby considerable vacuolation. Intine development begins just prior to pollen grain mitosisand continues rapidly at the aperture. The thin foot layer becomesdiscontinuous. Further intine deposition takes place after mitosisand a bilayer is apparent in mature grains. The matrix of thislayer contains conspicuous electron-opaque platelets. The exineof the mature spore stains less intensely than in the youngspore and the interbacula spaces are filled with material fromthe degenerate tapetum. Gibasis karwinskyana, Gibasis venustula, Commelinaceae, exine, intine, tapetum, pollen wall, ultrastructure     

Pollination, Pollen Viability and Pistil Receptivity in Cucurbita pepo

Cucurbita pepo carries male and female flowers on the same plant,and is pollinated by nectar-collecting bees. The nectaries aredimorphic in the two sexes and pollen is loaded and unloadedas the bees gain access to the nectar. Both types of flowerare open for only 6 h (from 0600 h to 1200 h); male flowersopen and close half an hour earlier than female flowers. Thelatter produce more nectar and are visited more often by thebees than the male flowers. Pollen viability determined by fluoresceindiacetate (fluorochromatic reaction) decreases by 20% duringanthesis and more rapidly after the flower closes. This decreaseis due to dehydration of the grain, especially around the porewhere the intine is exposed. An unusual feature of this speciesis that the grains do not dehydrate before anther dehiscence.Female receptivity has two aspects, that of the stigma lasting4 d, and that of the ovules lasting 2 d. The receptivity ofthe two sexes and the short period of anthesis are discussedin the light of the reproductive ecology of the species.Copyright1993, 1999 Academic Press Entomophilous pollination, anthesis, nectars, pollen viability, female receptivity, bees, pollinator efficiency, courgette Cucurbita pepo, Cucurbitaceae     

Efficiency of Countercurrent Distribution, Sephadex G-10, and Silicic Acid Partition Chromatography in the Purification and Separation of Gibberellin-like Substances from Plant Tissue

The effectiveness of countercurrent distribution, Sephadex G-10column chromatography and silicic acid partition column chromatographyin the purification of gibberellin-like substances from extractsof etiolated Phaseoltu multiflorus seedlings, and elongatingvegetative shoots of Arizona cypress (Cupressus arizonica Greene)and coastal Douglas-fir (Pseudotsuga menziesii var. menziesii)was followed by the use of the barley half-seed -amylase bioassayand dry-weight measurements. Countercurrent distribution resultedin a 10- to 30-fold reduction in the dry weight of the acidic,ethyl acetate-soluble fraction. Sephadex G-10 column chromatographyfurther lowered the dry weight by about two-thirds. Silicicacid partition column chromatography separated gibberellin-likesubstances from each other and again reduced the dry weight.Enhancement of bioassay activity was noted at each step of thepurification procedure. It is concluded that the above proceduresconstitute useful and efficient tools for the initial purificationof gibberellin-like substances from plant tissue.     

Tapetal Cell Changes and Sporoderm Development in Rhigozum trichotomum (Burch.)

Rhigozum trichotomum is a perrenial woody shrub which is indigenousto the arid regions of southern Africa. Primexine developmentis initiated while the microspores are still enclosed by callose.This is followed by the appearance of probacula which give riseto the tectum, bacula and nexine. At the time of callose dissolution,the exine pattern is well established and intine developmenthas been initiated. During the tetrad stage, the protoplastsof the tapetal cells exhibit shrinkage while conspicuous stacksof rough endoplasmic reticulum become evident in their cytoplasm.These stacks produce numerous vesicles which are associatedwith lipid globules and which migrate to the tapetal/locularwall where, it is suggested, they give rise to the pro-orbicules.The pro-orbicules become coated with an osmiophilic substance,probably sporopollenin, and are released into the thecal fluidto become intimately bound to the exine, Here they are strippedof the osmiophilic layers which appear to be incorporated intothe sporoderm. Rhigozum trichotomum (Burch.), sporoderm, pollen wall, exine, orbicules, pro-orbicules, sporopollenin, tapetum     

Preparation of Chlorophyll a, Chlorophyll b and Bacteriochlorophyll a by Column Chromatography with DEAE-Sepharose CL-6B and Sepharose CL-6B

Chlorophylls extracted from spinach leaves were made free fromcarotenoids, pheophytins, chlorophyllides and plastoquinonesby column chromatography with DEAE-Sepharose CL-6B. Chlorophylla and b were separated by column chromatography with SepharoseCL-6B. By a combined use of DEAE-Sepharose CL-6B and SepharoseCL-6B, pure chlorophyll a and b were prepared from the leavesin a short time. Bacteriochlorophyll a_p extracted from a photosyntheticbacterium Chromatium vinosum was made free from carotenoids,bacteriopheophytin, ubiquinone and lipids by column chromatographywith Sepharose CL-6B. (Received April 19, 1983; Accepted June 16, 1983)     

Periodicity of Pollen Development and Quantitative Cytochemistry of Exine and Intine Enzymes in the Grasses Lolium perenne L. and Phalaris tuberosa L.

Daily analysis of anther samples during flower development hasenabled an estimation of the duration of defined developmentalperiods in pollen of the grass Phalaris tuberosa. A similarsequence of pollen development has been established for ryegrass,Lolium perenne, where changes in activity of wall enzymes havebeen followed using quantitative cytochemical methods. Acidphosphatase, an intine enzyme, showed two periods of activity:during the vacuolate period corresponding to deposition of theintine polysaccharides; and in the maturation period correspondingto cytoplasmic activity. Non-specific esterase showed greatestactivity in the parietal tapetal cells until their dissolutionearly in the vacuolate period when an increase in pollen-associatedactivity occurred. These changes provide additional evidencefor the transfer of tapetal proteins to exine sites. Lolium perenne L., Phalaris tuberosa L., ryegrass, canary grass, pollen development, quantitative cytochemistry, enzyme activities, acid phosphatase, esterase     

Foraging behavior of the predaceous cladoceran, Leptodora kindti, and escape responses of their prey

Using silhouette video photography we have made the first quantitativeobservations of foraging behavior in Leptodora kindti, a predaceouscladoceran (Haplopoda). Leptodora swims with a mean velocityof 13.4?4.0 mm s^–1 and initiates an attack only upon directcontact with potential prey. The attack sequence is as follows:Leptodora swims randomly through the water column with all fivepairs of thorac appendages spread to form a ‘feeding basket’and, seemingly by chance, encounters prey. Shortly after preymake contact with any part of Leptodora's body (usually ventral),the abdomen is rapidly pulled forward, clamping itself underthe feeding basket so that the telson closes it at the posteriorend. The duration of this movement is always the same and weconclude that it is an indiscriminate reflex. If the prey isencountered anywhere but a short distance directly in frontand slightly below the Leptodora, it is not captured. The speedof copepod escape responses effectively allows them to avoidcontact with the predator. Daphnia's escape response, particularlythat of juveniles, is slower and leaves them far more susceptibleto Leptodora predation.     

Purification and Characterization of an Aminopeptidase, LPAase 2, from Euonymus Leaves

An aminopeptidase, LPAase 2, from the leaves of Euonymus alatusf. ciliato-dentatus was purified about 240-fold by a combinationof DEAE-cellulose and Sephadex G-100 column chromatographies.The molecular weight of LPAase 2 was estimated to be about 62,000,and the optimum pH for the hydrolytic activity against leucinep-nitroanilide(LPA) was 7.6. LPAase 2 hydrolyzed LPA, leucine-rß-naphthylamide(leucine-NA), phenylalanine-NA and tyrosine-NA. It was inhibitedstrongly by p-chloromercuribenzoate (PCMB), iodoacetic acidand heavy metal ions, but was not affected by thiol compoundsand metal-chelating reagents. Therefore, a sulfhydryl groupcould be involved in the active site of LPAase 2. None of themetal ions tested promoted LPAase 2 activity. The propertiesof LPAase 2 were compared with those of aminopeptidases reportedfor other plants. (Received November 24, 1983; Accepted April 16, 1984)     

Effects of ovular secretions on pollen in Pseudotsuga menziesii (Pinaceae)

Effects of ovular secretions on pollen grains were examined in Pseudotsuga menziesii. The exine is cast off in the micropylar canal. A membranelike structure covers parts of pollen grains and appears to protect them. The outer intine consists of fibrous materials, but it also shows a thicker filamentous appearance in some ovules during pollen elongation. The inner intine is electron-dense. Its fibrous nature is occasionally visible. Dissolution of the outer intine varies in amount and manner in ovules from different trees. The plasma membrane near the pollen wall alternatively appears normal and distorted. These different morphologies of the outer intine and of the plasma membrane are considered to result from secretions from the ovule. The outer intine may contain electron-dense globules that are formed in the tube cell and traverse the inner intine. Pollen tube formation appears to be triggered by a secretion from the ovule. Cross-pollinated grains are less distorted compared with self-pollinated grains.     

The diel migrations and vertical distributions of zooplankton and micronekton in the Northwestern Mediterranean Sea. 1. Euphausiids, mysids, decapods and fishes

Vertical distributions and diel migrations of the main speciesof micronekton, four euphausiids, one mysid, one decapod andthree fishes, were described in detail in the 0–1000 mwater column on a fixed station in the Northwestern MediterraneanSea. The euphausiids Euphausia krohni and Thysanopoda aequalis,the decapod Gennadas elegans and, to a lesser extent, the fishArgyropelecus hemigymnus were shown to perform clear diel verticalmigrations. Results of horizontal hauls at a given depth aroundsunrise and sunset showed a marked diurnal symmetry of the migratorycycles, particularly for E.krohni, T.aequalis and G.elegans.The behaviour of the euphausiid Nematoscelis megalops was morecomplex: it presented a repetitive bimodal day distributionand only part of its population migrated. As very weak or non-migratorswe found the euphausiid Stylocheiron longicorne and the bathypelagicmysid Eucopia unguiculata, for which migration concerned onlysome of the older individuals. The fishes Cyclothone braueriand Cyclothone pygmaea appeared to be non-migrants. As depthincreased, C.braueri was replaced by C.pygmaea, with maximumconcentrations at 350–550 and 550–700 m depth, respectively.     

Occurrence of dinoflagellate Alexandrium tamarense, a causative organism of paralytic shellfish poisoning in Chinhae Bay, Korea

A paralytic shellfish poisoning (PSP) incident caused by consumptionof the mussel Mytilus edulis occurred for the first time inKorea in April 1986. Weekly water samplings were carried Outduring the period from 7 March to 21 April 1989 in Chinhae Bay,Korea, in order to identify the causative organism. The temperaturecharacteristics of the water column indicated three differenthydrological regimes: well mixed (up to 7 March), weakly stratified(17–31 March) and stratified (7–21 April). Toxicityof the phytoplankton was detected during the weakly stratifiedperiod, but only in the 10–50 p.m phytoplankton size fraction.This study presents the occurrence of the toxigenic dinoflagellateAlexandrium tamarense, which is a causative organism of PSP,in Korean coastal waters. Its biomass varied at different depthsin the water column, ranging from 200 to 8000 cells 1^–1in the water column. The weekly fluctuation of A.tamarense toxicitywas similar to that of mussel toxicity. ¹ Present address: Department of Biology, College of NaturalSciences, Hanyang University, Seoul 133-791, Korea     

Pollen-wall Proteins: 'Gametophytic' and 'Sporophytic' Fractions in the Pollen Walls of the Malvaceae

Proteins are stored in two sites in the pollen grain walls ofthe Malvaceae, (a) in the cellulosic intine, mainly in the vicinityof the circular apertures, and (b) in cavities in the sculpturedlayer of the exine. The intine-held materials are incorporatedduring the growth of the wall. The exine materials are derivedfrom the tapetum, which during dissolution releases cistemaewith a granular-fibrillar content bounded by ribosomal membranes.This fraction is injected into the exine cavities after thecompletion of wall growth through micropores in the tectum.PAS-reacting material is associated with the injected protein.Another tapetal fraction, lipid in nature and commonly containingcarotenoids, remains on the surface of the pollen grains toform the Pollenkitt. While protein can be detected cytochemically in both intineand exine sites, acid phosphatase and ribonuclease activitywas found to be associated only with the former. Immunofluorescencemethods using antiserum to total pollen leachates showed thatantigens are present in both sites. When the pollen grains are moistened, the exine-held proteinsbegin to pass out through the micro-pores in the tectum within30 s of moistening, while the main discharge from the aperturalintine follows in 4–5 min. These observations, together with evidence from other families,suggest that the intine-held proteins of angiosperm pollen grainsare always produced by the male gametophyte, while those heldin exine cavities are sporophytic in origin, being derived fromthe tapetum. As previously proposed, it seems probable thatin intraspecific incompatibility systems of the gametophytictype control is mediated through intine-held ‘recognitionsubstances’, whereas in sporophytic systems the exine-heldmaterials are concerned.     

Seasonal changes in composition of the cyanobacterial community and the occurrence of hepatotoxic blooms in the eastern townships, Quebec, Canada

Four eutrophic lakes in the eastern townships (Québec,Canada) were sampled on a biweekly basis between May and October2001 to examine seasonal changes, and to study the role of taxonomicand environmental factors in cyanobacterial toxin production.Microcystin-LR (MC-LR) equivalent content was determined usinga protein phosphatase inhibition assay on extracts of lyophilizedplankton. Three of the lakes showed a similar pattern of maximumwater column toxicity in late summer, while in the fourth, toxicitywas highest in spring and then declined over the year. Variationsin water toxicity level could be attributed to the abundanceof two potentially toxigenic genera, Microcystis and Anabaena.A multiple regression model explained 75% of the variation inmicrocystin (MC) concentration, based on water column totalnitrogen concentration (TN) and the biomass of these two genera.Microcystis and Anabaena genera appeared to be similarly toxicin all lakes. Increased water column stability, higher lightextinction coefficient and a lack of dissolved nutrients wereall associated with increased total biomass of toxigenic cyanobacterialgenera.