Energy-driven Protein Release from Germinating Pollen of Petunia hybrida

The gradual release of proteins from Petunia hybrida pollenduring 5 h of germination in vitro is severely inhibited bythe uncouplers 2,4-dinitrophenol (DNP) and carbonylcyanide-m-chlorophenylhydrazonc(CCCP) as well as by the ATPase inhibitors N,N'-dicyclohexylcarbodiimde(DCCD) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).In the presence of these so-called energy poisons, a small amountof protein is given up to the culture medium in the first hourand no further release is seen. Cyclohexiinide, an inhibitorof protein synthesis, also inhibits much of the later gradualprotein export, and gel electrophoresis (SDS-PAGE) coupled withautoradiography suggests that a small fraction of the proteinsreleased is newly synthesized but they are different from mostof the major proteins exported to the medium. It is concludedthat in addition to the diffusible proteins passively exportedin the first hour, a major proportion of protein is releasedfrom the pollen by an energy-driven process, inhibited by thethiol reagent N-ethylmaleimide, during the 5 h of germination.A small number of these proteins is synthesized during pollengermination. Petunia hybrida L, pollen protein release, energy-dependent, germination, protein synthesis     

Detection of Pollen Germination Inhibitors in Brassica oleracea Tissue Extracts

Petunia hybrida and Lilium lankongense pollens were germinatedon thin layer chromatography (TLC) plates following chromatographyof extracts from the self-, cross- and unpollinated stigmas,styles and ovaries and the seeds, leaves and pollen of threeinbred Brassica oleracea families. Zones of pollen germinationinhibition on the TLC plates showed that inhibitory compoundswere present in the tissue extracts. The R_f values and numberof these compounds varied with the tissue used, stigma tissuecontaining the largest amounts and the greatest number of inhibitors.In contrast, differences between the inbred lines tested wereslight and quantitative. Pollen from both P. hybrida and L.lankongense gave the same results; that from B. oleracea couldnot be used because of its poor germination. Brassica oleracea, Brussels sprout, kale, Lilium lankongense, Petunia hybrida, pollen germination, thin layer chromatography, germination inhibitors, phytoalexin, bioassay     

Pollen Diffusates of Crotalaria retusa and their Role in pH Regulation

Details of the release of proteins and amino acids from culturedpollen grains and the role of the leached metabolites in pollengermination, pollen tube growth and regulation of pH of theculture medium in Crotalaria retusa have been investigated.In unbuffered media, satisfactory pollen germination and tubegrowth occurred over a wide range of pH values 4.0–9.0.This was related to the ability of pollen diffusates to shiftthe pH to 6.25 in all these media. Similar pollen germinationand pH shift was observed when the pollen was eluted twice beforeculturing. When the pH shift was reduced by using buffered media,optimal germination and tube growth occurred only at pH 6.0.Pollen diffusates had a strong buffering capacity. Proteinsand amino acids released from pollen do not seem to have a directrole in pH regulation. The components involved in pH regulationmay originate from the pollen wall as well as from the cytoplasm. Crotalaria retusa L, pH regulation, pollen diffusates, pollen germination     

Sugars in Natural and Artificial Pollen Germination Substrates

The stigmatic exudates and pollen grains of five unrelated specieswere tested for sugars. Glucose, fructose, and sucrose werefound in the stigmatic fluid of Yucca aloifolia L. and glucoseand fructose in that of Oenothera adrummondii Hook. In in vitroexperiments with Y. aloifolia pollen, high germination percentageswere obtained in artificial media containing glucose or sucrose.Fructose, which is present in the stigmatic fluid of the Yuccasp. resulted in high in vitro pollen germination only when borateand calcium were added to the medium. Presence of bound sugarsis indicated in the stigmatic secretion of Citrus aurantiumL. and pollen of the single plant tested germinated at a lowpercentage in artificial sugar media. No sugars were detectedin the stigmatic fluids of Hemerocallis fulva L. and Zea maysL. and in these two species in vitro pollen germination in sugarymedia was negligible or absent. The pollen grains of all five species contain sucrose and thoseof Oenothera and Citrus also reducing sugars.     

Biochemical and Ultrastructural Changes in Pollen ofZea maysL. Grown Under Enhanced UV-B Radiation

The influence of ultraviolet-B (UV-B) radiation on the developmentof the male gametophyte was studied inZea maysL. cv. LG12 grownin a growth chamber under PAR light supplemented with UV-B radiationand compared with a second set of plants grown under PAR light.Pollen samples collected from both groups of plants were culturedon germination medium and it was found that UV-B had no effecton pollen germination. Total pollen protein content was notaffected but UV-B absorbing pigments increased. Some ultrastructuralalterations were observed in pollen and pollen tubes, in particularlarge amounts of electron dense deposits were seen throughoutthe cytoplasm and in association with the pollen wall. In maturespikes of UV-B treated plants, anthers retained numerous pollengrains in their loculi while anthers of control plants werealmost empty. UV-B treatment delayed flowering by 2–3d. These results show that UV-B treatment of maize plants interfereswith flowering, pollen ultrastructure and anther maturationeven though pollen germination is unaffected. The significantincrease of UV-B absorbing pigments in pollen grains could representa defence mechanism that enables plants to complete their reproductivecycle.Copyright 1998 Annals of Botany Company Zea maysL., maize, UV-B radiation, pollen.     

The Effect of Brassica oleracea Stigma Extracts on the Germination of B. oleracea Pollen in a Thin Layer Chromatographic Bioassay

Hodgkin, T. and Lyon, G. D 1986. The effect of Brassica oleraceastigma extracts on the germination of B. oleracea pollen ina thin layer chromatographic bioassay.—J. exp. Bot. 37:406–411. A procedure for germinating Brassica oleracea pollen on thinlayer chromatography plates pretreated with 20 mol m^–3tris(hydroxymethyl) methyl-aminopropanesulphonic acid (TAPS)buffer, pH 8·0 has been devised and used to detect pollengermination inhibitors in B. oleracea stigma extracts. Inhibitory zones in extracts of stigmas, unpollinated, or collected0·5, 4, 8 and 24 h after self- or cross-pollination,differed little in R_F values and sizes. Extracts of stigmascollected 1 h and 2 h after self-pollination gave a small additionalinhibitory zone which was not detected in 1 h and 2 h cross-pollinatedstigma extracts. The results showed some differences from thoseobtained using Petunia hybrida pollen germinated on T.L.C. platesthat were not pretreated with buffer. The nature of the differencesbetween the two bioassays is discussed and some possible reasonsfor them indicated. Key words: Pollen, germination inhibitors, self-incompatibility, Brassica oleracea     

Sensitivity to Low Temperature in Pollen Germination and Fruit-Set in Cicer arietinum L.

Cicer arietinum L. cv. G 62404 was seen to have a very low fruit-setpercentage at low temperatures and as temperatures increased,either in nature or in culture conditions, the fruit-set alsoincreased. This effect of temperature could also be seen inpercent pollen germination and pollen tube growth. M 450, amutant of G 62404, was seen to have a better fruit-set percentageat low temperature and better pollen germination. This may bedue to differences in malic acid concentration, which increasedpollen germination, and pollen tube growth under in vitro cultureconditions.     

Viability and Germination of the Pollen of Sorghum [Sorghum bicolor (L.) Moench]

Previous studies have demonstrated that pollen of sorghum [Sorghumbicolor (L.) Moench] loses capacity to both germinate in vitroand to set seed in vivo soon after being shed. The current studyevaluates the capacity for dehydrated pollen to effect in vitrogermination, reduce tetrazolium chloride, and set seed on cytoplasmicmale sterile plants. Morphological changes during pollen germinationwere examined by scanning electron microscopy (SEM). Close to70% of the pollen germinated in 5 min, or less, when collectedat 80% relative humidity (RH) and stored in sealed glass vials.Pollen tubes elongated autotropically with atmospheric humidityapparently being a controlling factor in the process. Pollendehydrated at 50% RH and 25°C for 15-30 min neither germinatedin vitro, reduced tetrazolium chloride, nor set seed on malesterile plants. Rehydrating the pollen did not restore the capacityfor germination. SEM micrographs demonstrated that elongatingpollen tubes encircled the pollen grain and were contiguousto the surface. A fibrillar-like material existed on the exineof separated pollen grains at the point where the grains hadbeen previously attached.Copyright 1994, 1999 Academic Press Sorghum pollen, germination, seed-set, viability, scanning electron microscopy, Sorghum bicolor (L.) Moench     

Membrane State and Pollen Viability

The relationship between germinability and fluorochromasia (FCR)has been studied in pollen of eight genera, Secale, Iris, Carex,Eleocharis, Cytisus, Digitalis, Plantago and Lonicera. The FCRtests two properties of the pollen, (a) the integrity of theplasmalemma of the vegetative cell and (b) the presence of anesterase capable of cleaving the fluorogenic ester, fluoresceindiacetate. In general, the correlations between FCR and germinabilitywere found to be very highly significant. This is interpretedas meaning that the primary determinant of pollen viabilityin short-term storage is the state of the vegetative cell membranes.It is suggested that in the partly dehydrated grain at the timeof dispersal the membranes are largely dissociated and do notform an osmotic barrier, but that normal properties are recoveredduring controlled hydration which would normally take placeon the stigma. According to this view, the decay of apparentviability is related to the progressive loss of the capacityof the vegetative cell membranes to regain a normal structureon rehydration. The genera investigated varied in their longevityin storage in low and high humidity conditions. After low-humiditystorage, most showed some enhancement of germination with 1h exposure to a humid atmosphere before transfer to the germinationmedium. During this ‘conditioning’ period the membranesrecover their fluorescein retentivity in step with the increasein germinability. pollen testing, pollen membranes, pollen fluorochromasia, Secale cereale L., Iris pseudacorus L., Carex ovalis Good., Carex nigra (L.) Reichard, Eleocharis palustris (L.) R.Br., Cytisus battandieri Maire, Plantago lanceolata L., Digitalis purpurea L., Lonicera periclymenum L.     

Pollen Tube Development and Characteristics of the Protein Emission in Conifers

When ripe, viable pollen of Pinus contorta, Pinus nigra, Piceasitchensis, Abies alba and Cedrus deodara is germinated on asuitable artificial substrate, the process of pollen tube growthbegins 12–36 h later, according to the species. The tubeemerges at the leptoma, a structurally specialized site in thepollen wall, and the early tube wall is continuous with thetwo microfibrillar intine strata within the grain. Later inpollen tube development, when cytoplasmic zonation has beenestablished, only the inner of these two wall layers is differentiated. Cytochemical methods show that, during hydration and throughoutthe period of tube growth in culture, proteins are releasedfrom the pollen grains; before germination most conspicuouslyfrom the leptoma, subsequently from the tube itself. The emissioncontains a number of hydrolytic enzymes and a non-catalyticmoiety. Gel-electrofocusing reveals that the hydrolases releasedfrom germinating Pinus contorta pollen include several acidphosphatase and esterase isozymes, and that there are differencesbetween the composition of the emission and the spectrum ofsoluble proteins extracted from the pollen grains before germination.Analysis by immunodiffusion demonstrates that two componentswith antigenic characteristics present in the quiescent pollengrains are represented in the emission. The possible utilityof the released components in the biology of conifer reproductionis discussed. Pinus contorta, Pinus nigra, Picea sitchensis, Abies alba, Cedrus deodara, lodgepole pine, Austrian pine, Sitka spruce, European silver fir, deodar, pollen tube, pollen germination, protein release, isoelectric focusing     

RNA and Protein Synthesis in Germinating Pine Pollen

The results of autoradiographic experiments demonstrate that,as with the pollen of most other species, both the generativeand vegetative nuclei of Loblolly Pine (Pinus taeda) activelyengage in RNA synthesis from the very early stages of pollengermination. Unlike most other species, however, this newlysynthesized RNA includes rRNA. Evidence is provided for theimportance of this newly synthesized RNA in the process of continuedpollen tube growth. One and two-dimensional gel electrophoretic analysis revealsa number of both qualitative and quantitative differences amongthe proteins synthesized during the early stages of germinationand the later stages of pollen tube growth. One of the mostnotable of these is a 36 kD protein, the synthesis of whichpredominates during the later stages of pollen germination.A similar pattern of 36 kD protein synthesis is observed whenmRNA extracted from pollen at each of these stages is translatedin vitro. Key words: Pinus, pollen tube growth     

Papillate Tepal Hoods and Delayed Pollen Germination inAlbucaL. (Liliaceae)

In the genusAlbuca, pollen germination is delayed until thetepals close around the stigma immediately before the onsetof floral senescence. At this time, papillae on the tepal apicesand the stigma swell and produce an exudate in which pollenrapidly germinates. No pollen germination occurs when the tepalsare artificially prevented from closing around the stigma.Copyright1998 Annals of Botany Company Albuca, Liliaceae, delayed pollen germination, gametophytic self-incompatibility, perianth contributing to pollen germination, pollen tube inhibition in ovary.     

Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity througha sequence of developmental stages of the pistil have been studiedin four taxa characterized by having wet stigmas — Petuniahybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata.The style is solid in the first two and hollow in the lattertwo taxa. In all the taxa, stigma—surface esterase couldbe detected in a thin surface layer (pellicle) from a very earlystage of pistil development, irrespective of the presence orabsence of the exudate. However, the taxa showed variation instigma receptivity. In Petunia and Nicotiana, stigmas from pistilsof all the stages supported pollen germination and tube growth.In Amaryllis and Crinum, stigmas of only the mature pistils,when the exudate is present on the stigma, supported normalpollen germination and tube growth. It is inferred that in taxacharacterized by a wet stigma and solid style, the factors requiredfor pollen germination are present from an early stage of pistildevelopment and the exudate per se is not involved in pollengermination. In taxa characterized by a wet stigma and hollowstyle, however, the pellicle does not carry the factors requiredfor pollen germination and tube growth; they appear to be presentin the exudate. Petunia hybrida Vilm, Nicotiana tabacum L., Crinum defixum, Ker-Gawl, Amaryllis vittata Ait., tobacco, pollination, pollen germination, stigmatic exudate, stigma receptivity, stigma-surface esterase, esterase activity     

GA_3对含笑花粉萌发及花粉管生长的影响

以含笑（Michelia figo）花粉为试材,采用花粉离体培养法,研究GA3对含笑花粉萌发和花粉管生长的影响。结果表明,GA3可以促进含笑花粉提早萌发,20～200 mg/L GA3对含笑花粉萌发和花粉管生长起促进作用,浓度超过200 mg/L花粉萌发和花粉管生长均受到抑制。以GA3200 mg/L的促进作用最好。     

Pollen DNA repair after treatment with the mutagens 4-nitroquinoline-1-oxide, ultraviolet and near-ultraviolet irradiation, and boron dependence of rapair

Summary Irradiation of dry, mature pollen from Petunia hybrida with near-ultraviolet light from an erythemal-sunlamp gave rise to a repair-like, unscheduled DNA synthesis during the early stages of in vitro germination. Like that brought about by farultraviolet light from a germicidal lamp, this DNA synthesis is enhanced by hydroxyurea added to the germination medium, and reduced by photoreactivating light given after ultraviolet irradiation and before germination begins. It is concluded that pollen, often receiving considerable exposure to sunlight, has, in addition to the protection afforded by the ultraviolet filtering effect of yellow pigments, also the capacity to repair ultraviolet produced changes in DNA, by both photoreactivation and dark repair processes.Because mature Petunia pollen is arrested at the G₂ stage of the cell cycle, germinating pollen provides us with a highly synchronous plant tissue with a very low background of DNA replicative synthesis suitable for sensitive measurement of DNA repair synthesis. Thus we have shown that 4-nitroquinoline-1-oxide, at concentrations greater than 0.001 mM, gives rise to an unscheduled DNA synthesis which is enhanced by hydroxyurea. Like that induced by ultraviolet radiation, the chemical mutagen brings about DNA repair only during the early stages of pollen germination, and further it has been possible to show that repair ceases at about the time that generative cell division and pollen tube elongation begins.Boron addition enhances both ultraviolet and 4-nitroquinoline-1-oxide induced repair synthesis. By delaying the chemical mutagen initiation of repair until after germination has begun, we have been able to show that boron is most beneficial during the first hour of germination. It is postulated that this is achieved through an as yet unknown effect of boron on the supply of precursors before pollen cell metabolism is fully committed to pollen tube synthesis later in the germination period.     

GA3对含笑花粉萌发及花粉管生长的影响

以含笑(Michelia figo)花粉为试材,采用花粉离体培养法,研究GA3对含笑花粉萌发和花粉管生长的影响。结果表明,GA3可以促进含笑花粉提早萌发,20～200 mg/L GA3对含笑花粉萌发和花粉管生长起促进作用,浓度超过200 mg/L花粉萌发和花粉管生长均受到抑制。以GA3200 mg/L的促进作用最好。     

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     

The Comportment of the Vegetative Nucleus and Generative Cell in the Pollen and Pollen Tubes of Helleborus foetidus L

It has been reported that in species of Plumbaginaceae, Chenopodiaceae,Cruciferae and Amaryllidaceae a ‘male germ unit’is formed in which the two male gametes remain inter-connected,with one of the pair linked intimately to the vegetative nucleus.In two species the unit has been shown to remain intact in thepollen tube, and some accounts imply that it is polarized inits movement, the vegetative nucleus leading in the tube. Evidence given in this paper indicates that such a unit is unlikelyto be present in Helleborus foetidus L. (Ranunculaceae). Applicationof an optical sectioning technique has shown that at no timeis there a persistent linkage between the generative cell andthe vegetative nucleus in unhydrated, hydrated and germinatingpollen, nor is one present in the early pollen tube. Furthermore,no inter-connections between the two entities were seen in protoplastsfrom living, hydrated and incipiently germinating grains isolatedmechanically in an osmotically balancing medium. Following germination,the vegetative nucleus leaves the grain in advance of the generativecell in most instances, but in the samples examined the generativecell led in about 30 per cent of the tubes. Assembling a polarisedmale germ unit in these circumstances would require (a) theformation of an inter-connection between the vegetative nucleusand the generative cell or one of the gametes derived from itduring passage through the tube, and (b) where the generativecell initially leads in the tube, an exchange in relative positions.It is considered improbable that these conditions could consistentlybe met. Mature, incipiently germinating pollen of H. foetidus releasesa fibrillar component when extruded into suitable media. Websor clusters of fibrils are commonly seen to be associated withboth the vegetative nucleus and the generative cell. The possibilitythat the fibrils are composed of aggregates of microfilamentsis considered. Helleborus foetidus L., pollen germination, generative cell, vegetative nucleus, male germ unit     

Studies on the Initial Biochemical Reactions in the Germination of Pollen Grains

Tritiated water (³HHO) has been used as the medium for the germinationof pollen grains of Pinus radiata D.Don (a gymnosperm), Ulexeuropaeus L., Salix caprea L. (dicotyledonous angiosperms),and Phormium tenax Forst. (a monocotyledonous angiosperm). ³H-labelledcompounds formed during the initial germination (egersis) periodhave been separated and identified to give information on themetabolism taking place.Since the earliest labelled compoundswere -aminobutyric acid, alanine, aspartic acid, and glutamicacid, it is concluded that these amino acids play an importantpart in the biochemical reactions during the early stages ofgermination. Citric acid, malic acid, and glutamine do not becomelabelled until later while carbohydrates, phosphate esters,and lipids do not appear to incorporate tritium within the firsthour of germination.The gross labelling pattern and the natureof the individually labelled metabolites, their intensity andsequence of labelling are similar for all the species investigatedexcept gorse, which showed a decrease in the intensity and thenumber of labelled metabolites with time.P. radiata pollen storedfor 3 years under vacuum or carbon dioxide has a labelling patternsimilar to freshly collected P. radiata pollen, except for aconsiderable increase in the amount of an unknown metabolite(Compound X).     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。