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PubMed2002

Bacillus megaterium spore germination is influenced by inoculum size.

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.

Bacillus megaterium↗
PubMed2002

A rapid staining technique for the detection of the initiation of germination of bacterial spores.

AIMS: We propose to apply the Wirtz-Conklin staining technique to evaluate spore germination. METHODS AND RESULTS: Spores at different stages of germination were stained with modified spore stain (Wirtz-Conklin) and evaluated for staining properties. Bacillus spores suspended in deionized water, which does not support germination, stained greenish-blue. Spores suspended in germination enhancers that did not form bacilli stained pink, indicating the initiation of germination. Spores suspended in culture media, which promotes bacterial outgrowth, formed bacilli and were also stained pink. CONCLUSIONS: Modified spore stain (Wirtz-Conklin) was found to be useful to detect the initiation of spore germination as early as 30 min following incubation in a germination environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple staining procedure is useful in detecting the initiation of germination of bacterial spores.

Bacillus↗
PubMed1980

Regulation of protein synthesis during spore germination in Dictyostelium discoideum.

Spore germination in the slime mold Dictyostelium discoideum is a particularly suitable paradigm for studying the regulation of gene expression because developmentally regulated changes in both protein and mRNA synthesis occur during the synchronous transition from dormant spore to growing ameba. To investigate the regulation of protein synthesis during germination, we labeled activated spores with [35S]methionine at 1-hr intervals during germination, until amebas emerged (at 3 hr). The labeled proteins were resolved by two-dimensional polyacrylamide gel electrophoresis. Six classes of proteins were distinguished, depending on the time of onset and duration of their synthesis: (i) proteins made only during the first hour of germination, (ii) proteins made during the second hour, (iii) proteins made during the third hour, (iv) those synthesized only between 1 and 3 hr after activation, (v) peptides made only between 0 and 2 hr after activation, and (vi) proteins that were made throughout germination, mRNA isolated from dormant spores and from spores at different stages of germination was translated in a wheat germ cell-free protein-synthesizing system, and the proteins made in vitro were compared to those synthesized in vivo. The majority of the changes in the pattern of protein synthesis that occurred during the different stages of germination were attributable to the presence or absence of translatable mRNA. It is concluded that the synthesis of a majority of the proteins during spore germination is transcriptionally controlled.

Cell-Free System↗
PubMed2003

Individual and coupled effects of echinoderm extracts and surface hydrophobicity on spore settlement and germination in the brown alga Hincksia irregularis.

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30 min were > 400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were > 300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30 min and the settled spores allowed to subsequently germinate for 24 h. Spore germling numbers were consistently > 25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24 h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30 min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24 h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.

Analysis of Variance↗
PubMed1969

Localization of immunological complexes fixing beta1C (C3) in germinal centers of lymph nodes.

28 human and 60 experimentally stimulated rabbit lymph nodes were studied by means of light microscopy and immunofluorescence. 21 of the 28 human lymph nodes showed well-developed germinal centers. IgM, IgG, and the beta(1C) component of complement were found in the same distribution within germinal centers when examined in serial cryostat sections. 36 rabbits were stimulated with Brucella antigen, and 24 rabbits with BSA. A strikingly consistent correlation between distribution and appearance of specific staining for rabbit beta(1C), IgM, and IgG was observed; when lymph nodes were stimulated with BSA, antigen and specific antibody were present. Treatment of unfixed sections with citrate-buffered saline at low pH resulted in complete elution of immunoglobulins, beta(1C), and BSA from rabbit germinal centers, and in marked diminution of IgG and IgM in human germinal centers, while at the same time plasma cells remained strongly fluorescent. Specific selective fixation of heterologous (human) complement in rabbit germinal centers positive for beta(1C), IgG, IgM, and BSA was also obtained. These data present strong evidence for the existence within germinal centers of antigen-antibody complexes which fix at least the beta(1C) component of complement in vivo. The possibility of complete elution of immunoglobulins from rabbit germinal centers can be taken as evidence that, at least for 20 days after primary and secondary stimulation, a major component of the immunoglobulins present in germinal centers is not produced locally but accumulates at the surface of cells.

Animals↗
PubMed2002

Localization of the fertilized germinal disc in the chicken egg before incubation.

At the time of oviposition, the embryo in the fertilized chicken egg has developed the germinal disc on top of the yolk and contains thousands of blastodermal cells. The germinal disc escapes exact localization because it is surrounded by albumen and shell membranes under a calcified shell. This study investigated whether or not ultrasonography or nuclear magnetic resonance imaging (MRI) are suited to localize the germinal disc and, if so, to estimate the accuracy of localization. Hatchability of treated eggs was also recorded. The ultrasound waves were reflected by the shell. The germinal disc could be seen only after removal of the eggshell and the outer shell membrane. In all, 39 intact eggs, MRI localized the germinal disc within 2 mm thick image slices. The mean position of the germinal disc deviated 3.0+/-2.1 mm from the maximum vertical plane of the egg with a mean distance to the inner surface of the shell of 2.7+/-1.1 mm. Incubation resulted in 84.2 and 77.5% hatched chicks for imaged eggs and controls, respectively. The localization results of MRI were verified manually in 38 open shell cultures. Correlation coefficients for the position of the germinal disc were r = 0.86 to 0.99 for the x-axis, r = 0.75 to 0.89 for the y-axis, and r = 0.63 to 0.76 for the z-axis. The study thus shows that MRI is a reliable tool to localize the germinal disc within the intact freshly laid egg.

Animals↗
PubMed1991

Cellular desiccation and hydration: developmentally regulated proteins, and the maturation and germination of seed embryos.

Little more than a decade ago, 2-dimensional mapping of proteins and biochemical study of their allied coding elements (mRNA and DNA) were first used to probe possible changes in the embryo during seed germination. Because specification was of primary importance, our attention was initially directed toward the characterization of individual proteins and coding elements which, in preliminary surveys of the germinating wheat embryo, were found to be conspicuously subject to developmental regulation. Three of the proteins have become subjects of comprehensive investigations in this and other laboratories: the Em protein, the Ec protein, and germin. The Em and Ec proteins are encoded by the conserved mRNA 'stored' in the mature embryos of dry, field-ripened seeds but germin is encoded by the nascent mRNA formed after mature embryos are germinated in water. The Ec protein is the only bona fide Zn metallothionein yet found in higher plants. Studies of their biology and molecular biology suggest that the Em protein has a role in hormone-mediated (abscisic acid) cellular desiccation and that germin has a role in hormone-mediated (auxin) cellular hydration. It is projected that further studies of the Em protein may help elucidate the molecular basis for a loss of dessication tolerance during germination, and that further studies of germin may help elucidate the molecular basis of plant cell enlargement.

Cell Division↗
PubMed1991

Structural and stereospecific requirements for the nucleoside-triggered germination of Bacillus cereus spores.

A selection of adenosine analogues was tested for their ability to trigger germination of Bacillus cereus NCIB 8122 spores. The germination-inducing activity was governed by the structural properties of the sugar rather than the base moieties of the nucleosides. Among the sugar-modified analogues, only those containing a 2'-deoxy-D-ribose moiety promoted spore germination. Requirements for a specific molecular structure of the base were not clearly identified, although the highest activity was observed when substituents were inserted at position 6 of the purine ring. All the base-modified analogues, even those such as coformycin and 2'-deoxycoformycin with an expanded base ring, retained the germination-inducing activity of adenosine. However, of the two 2'-deoxycoformycin diastereoisomers characterized by an asymmetric carbon atom at position 8 of the homopurine ring, only the 8S-isomer induced germination, thus indicating that stereospecific configuration of the inducer, at least in the case of 2'-deoxycoformycin, appears to be essential for the initiation of spore germination. The differences in the germination-inducing activity of the various analogues tested were not affected significantly by spore activation at different temperatures, although the higher the activation temperature, the lower was the concentration of each analogue required for maximum germination.

Adenosine↗
PubMed1995

Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability.

The insect pathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (aw) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0.05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951-0.935 aw (identical to 95.1-93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 aw (identical to 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930-0.920 aw). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.

Culture Media↗
PubMed2006

Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea.

To analyse the role of trehalose as stress protectant and carbon storage compound in the grey mould fungus Botrytis cinerea, mutants defective in trehalose-6-phosphate synthase (TPS1) and neutral trehalase (TRE1) were constructed. The Deltatps1 mutant was unable to synthesize trehalose, whereas the Deltatre1 mutant showed elevated trehalose levels compared to the wild-type and was unable to mobilize trehalose during conidial germination. Both mutants showed normal vegetative growth and were not affected in plant pathogenicity. Growth of the Deltatps1 mutant was more heat sensitive compared to the wild-type. Similarly, Deltatps1 conidia showed a shorter survival under heat stress, and their viability at moderate temperatures was strongly reduced. In germinating wild-type conidia, rapid trehalose degradation occurred only when germination was induced in the presence of nutrients. In contrast, little trehalose breakdown was observed during germination on hydrophobic surfaces in water. Here, addition of cAMP to conidia induced trehalose mobilization and accelerated the germination process, probably by activation of TRE1. In accordance with these data, both mutants showed germination defects only in the presence of sugars but not on hydrophobic surfaces in the absence of nutrients. The data indicate that in B. cinerea trehalose serves as a stress protectant, and also as a significant but not essential carbon source for germination when external nutrients are low. In addition, evidence was obtained that trehalose 6-phosphate plays a role as a regulator of glycolysis during germination.

Adaptation, Physiological↗
PubMed2001

Release of reactive oxygen intermediates (superoxide radicals, hydrogen peroxide, and hydroxyl radicals) and peroxidase in germinating radish seeds controlled by light, gibberellin, and abscisic acid.

Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2',7'-dichlorofluorescin to fluorescent 2',7'-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens.

Abscisic Acid↗
PubMed1967

Periods of sensitivity to chilling in germinating cotton.

Cotton seedlings were subjected to a 96 hour chilling treatment (5 degrees or 10 degrees ) after periods of germination at 31 degrees ranging from 0 to 48 hours. Inhibition of subsequent growth at a favorable temperature by chilling was dependent on level of low temperature and stage of seedling development when chilled. Two periods of chilling hypersensitivity were observed during germination: 1) coincident with subjection of seed to a germination environment; and 2) after 18 to 30 hours of germination at 31 degrees . Subsequent growth of seedlings chilled after 12 to 18 hours or 48 hours of germination at 31 degrees was relatively unaffected. It is suggested that chilling alters specifically timed events that occur at the initiation of germination and after 18 to 30 hours of germination, and that alteration of these germination processes is visited on long term subsequent growth of the plant.

Journal Article↗
PubMed1969

Immunochemical Studies on Arachis hypogaea Proteins With Particular Reference to the Reserve Proteins. II. Protein Modification During Germination.

alpha-Conarachin and alpha-arachin are metabolized differently during germination. The amount of alpha-conarachin decreases without the appearance of antigenic intermediary compounds. alpha-Arachin retains its original antigenic structure and becomes more anodic during germination, but a number of intermediary proteins, differing widely in electrophoretic mobility, form up to 14 days germination. The electrophoretic shift of alpha-arachin, detectable after 1 day of germination, might be caused by progressive deamidation of glutamine and asparagine. Decreases in amide content of protein extracted after germination support the hypothesis and suggest that alpha-arachin provides the first source of ammonia to the new seedling.New antigenic proteins are synthesized in both roots and cotyledons during germination. Some appear organ specific, whereas others are identical in both the cotyledons and the roots. Appearance and disappearance of each new protein is unique with respect to period of germination and hence might correspond to specific events of germination.

Journal Article↗
PubMed1973

Photocontrol of the germination of onoclea spores: I. Action spectrum.

Light stimulates the germination of spores of the fern Onoclea sensibilis L. At high dosages, broad band red, far red, and blue light promote maximal germination. Maximal sensitivity to these spectral regions is attained from 6 to 48 hours of dark presoaking, and all induced rapid germination after a lag of 30 to 36 hours. Maximal germination is attained approximately 70 hours after irradiation. Dose response curves suggest log linearity. The action spectrum to cause 50% germination shows that spores are most sensitive to irradiation in the red region (620-680 nm) with an incident energy less than 1000 ergs cm(-2); sensitivity decreases towards both shorter and longer wavelengths. Although the action spectrum is suggestive of phytochrome involvement, photoreversibility of germination between red and far red light has not been demonstrated with Onoclea spores. An absorption spectrum of the intact spores reveals the presence of chlorophylls and carotenoids. Since the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea does not inhibit germination, it is concluded that photosynthesis does not play a role in the germination process.

Journal Article↗
PubMed1979

Germination-induced Changes in Chromosomal Proteins of Spring and Winter Wheat Embryos.

The template activity of chromatin from winter wheat embryos gradually increased during germination and was regulated with some nonhistone proteins different from the two major ones, molecular weight 39k and 59k polypeptides, previously reported.To clarify chromosomal proteins which are involved in regulation of template activity of chromatin, we studied the quantitative and qualitative changes in chromosomal proteins. Differences in acid-soluble and acid-insoluble proteins between chromatins from wheat germ and embryos germinated for various times were visualized by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.Nonhistone proteins of 39k, 41k, and 50k molecular weights were specifically present in wheat germ and in 24- or 48-hour germinated wheat embryos, thereafter greatly reduced or finally disappeared. In contrast, nonhistone protein of 37k was absent in germ and in embryos germinated for 24 hours and appeared after 48 hours of germination. Thereafter it was present in abundant amounts in 96-hour germinated winter wheat embryos and in 72-hour germinated spring embryos, corresponding to 7 and 10% of total nonhistone proteins, respectively. Histone H1, especially H1d, was slightly reduced after 48-hour germination, as much as basic nonhistone proteins having electrophoretic mobilities between H1d and H2B. Further-more, similarity and diversity of chromosomal proteins between spring and winter wheat embryos are shown in this study. A subspecies of histone H1c of spring wheat had faster electrophoretic mobility than that of winter wheat.

Journal Article↗
PubMed1983

Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature.

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25 degrees C, but not at 15 degrees C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15 degrees C and 25 degrees C.The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25 degrees C than at 15 degrees C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15 degrees C and 25 degrees C after the puncture force was reduced to between 0.3 and 0.4 newtons.Application of gibberellic acid(4+7) (100 microliters per liter) resulted in earlier germination at 15 degrees C and 25 degrees C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O(2) concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid(4+7), but only at 25 degrees C. At 15 degrees C, high O(2) concentrations slowed germination and endosperm strength decline.

Journal Article↗
PubMed1985

Germination, respiration, and adenylate energy charge of seeds at various oxygen partial pressures.

The effect of O(2) partial pressure on the germination and the respiration of 12 cultivated species was studied. The reciprocal of the time necessary to observe rootlet emergence in 50% of the seeds was used to approach the germination rate. The maximum germination and respiration rates were reached in most seeds at O(2) pressures close to that of air. Decreasing the O(2) pressure produced a gradual decrease of the germination rate. The seeds could be classed in two groups according to their response to low O(2) pressures. Group I includes lettuce, sunflower, radish, turnip, cabbage, flax, and soybean: at O(2) pressures close to 2 kilopascals, the germination in this group was stopped and the adenylate energy charge was lower than 0.6. Group II includes rice, wheat, maize, sorghum, and pea. The germination rate of these seeds was also gradually decreased by lowering the O(2) partial pressure but germination still occured, very slowly, at 0.1 kilopascal; the adenylate energy charge remained higher than 0.6. These differences in the germination rates and adenylate energy charge values could not be explained by differences in the sensitivity of respiration to O(2).

Journal Article↗
PubMed1987

Activities of isolated mitochondria and mitochondrial enzymes from aerobically and anaerobically germinated barnyard grass (echinochloa) seedlings.

Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O(2) uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a(3), and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists.

Journal Article↗