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In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts.

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.

Arabidopsis↗

[The influence of seed maturity and seed coat on the germination of Bupleurum chinense and B. falcatum].

OBJECTIVE: To investigate the factors influencing the seed germination of Bupleurum chinense and B. falcatum. METHOD: The seeds of different maturity, and the seeds whose coat were removed or nicked before dipping in water, were germinated in a light growth chamber. RESULT: The different inflorescence level represents different development period. Seeds harvested from cultivation have different maturity. The germination rate of the seeds in period I to III was nearly zero, and it was significant different from period IV to VII. The seed coat is not an obstacle for water absorption or gas exchange, but its removal led to significant increase of seed germination rate. However, to dip the seeds without coat into water for 48 h had no more effect on germination. CONCLUSION: The seed maturity and germination inhibitors in seed coat are two important factor influencing seed germination in B. chinense and B. falcatum.

Bupleurum↗

[Studies on germination characteristics of Prunella vulgaris seeds].

OBJECTIVE: To understand the effect of illumination, temperature on the germination of the Prunella vulgaris seeds. METHOD: The general germination method. RESULT: Illumination showed no clearly effect on the germination rate of the P. vulgaris seeds under proper temperature condition; However, illumination was the main factor to determine the germination rate of the P. vulgaris seeds when the temperature condition was unsuitable. The germination rate of the P. vulgaris seeds decreased with the time and increased when the marinating time was from 12 to 36 hours. When temperature reached 5-25 9 degrees C, the trend of the germination rate increased at frst and then decreased. CONCLUSION: The optimal germination condition of P. vulgaris seeds is 12 h of marinating time at the temperature of 20 degrees C under illumination.

Germination↗

[Effects of bryophytes in dark coniferous forest of Changbai Mountains on three conifers seed germination and seedling growth].

This paper studied the effects of Hylocomium splendens and Rhytidiadelphus triquetrus, the main bryophytes in dark coniferous forests of Changbai Mountains, on the seed germination and seedling growth of Pinus koraiensis, Picea koraiensis and Larix olgensis. The results indicated that at definite concentrations, the water extract of H. splendens inhibited Picea koraiensis seed germination, while that of R. triquetrus promoted it. Although the water extracts of the two bryophytes had no obvious effects on the seed germination of Picea koraiensis and Larix olgensis, they expedited the occurrence of the tree species' daily germination peak. The water extracts of test bryophytes inhibited the seedling growth of P. koraiensis and Picea koraiensis, but promoted that of Larix olgensis. The living shoots of the two bryophytes had no obvious effects on the seed germination of Picea koraiensis and Larix olgensis, but delayed the daily germination peak of Picea koraiensis while promoted that of Larix olgensis, andthe killed shoots inhibited the seed germination of all test tree species. Living shoots in larger amounts promoted the seedling growth of Picea koraiensis and Larix olgensis, but killed shoots were inadverse.

Bryophyta↗

[Study on characteristics of seed germination of Salvia officinalis].

OBJECTIVE: To investigate the influences of lightness and temperature on seeds germination of Salvia officinalis, and offer the basis for the standardized cultivation of S. officinalis. METHOD: The morphological characters, 1 000-grains weight and rate of water absorption of the seed were observed, the germination capacity, percent of germination power and germination index at the five different degrees: 10, 15, 20, 25, 30 degrees C were also measared. RESULT: 1 000-grains weight of the seeds was 8.03 g, the rate of water absorption is 49% within 24 h, the highest rate of germination capacity reached 85.33%, the germination index is 45.56. CONCLUSION: The lightness and 25 degrees C are the most suitable parameters for the seed germination of S. officinalis.

Germination↗

Confocal imaging and immunogold electron microscopy of changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L.

Using anti-myosin antibodies, standard immunocytochemical techniques in conjunction with confocal scanning laser microscopy and colloidal gold immunoelectron microscopy we compare changes in the distribution patterns of myosin during the early stages of pollen hydration, germination, tube growth, and myosin associated with isolated vegetative nucleus and the generative cell in Nicotiana tabacum L. Furthermore, on the Western blots of pollen tube proteins, the antimyosin antibodies crossreact only with one polypeptide of approximately 174 kDa. Confocal immunofluorescence microscopy reveals that in hydrated pollen, myosin is discretely associated with the cytoplasmic organelles and numerous punctate structures present in the center of the pollen. Within 30 min following transfer of pollen into the germination medium, that is, with the onset of germination, the centrally located punctate structures are displaced, and we find accumulation of myosin-associated organelles towards one of the germinal apertures from which the pollen tube would emerge. Subsequently, after 45 min of germination with the emergence of germination structure, few punctate structures are detected in the vegetative cytoplasm while intense immunostain is detected just below the plasma membrane of the emerging pollen tube tip. In the older parts of both short and long pollen tubes after 90 to 120 min of pollen germination, few fluorescent structures were found in the pollen tubes, however, numerous punctate fluorescent spots were concentrated in the tip region over a distance of 2 to 3 microns below the plasma membrane of the tube tip. This is further substantiated by colloidal gold immunoelectron microscopy wherein clusters of gold particles are associated with vesicle-like structures in the tip region of the pollen tubes.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗

The cyclic AMP/PKA signal pathway is required for initiation of spore germination in Schizosaccharomyces pombe.

Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.

Actins↗

Recombinant 25-kDa CD23 and interleukin 1 alpha promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis.

Germinal centers contain a proliferating pool of centroblasts which give rise to non-dividing centrocyte. Centrocytes are programmed to die by apoptosis unless they receive a positive signal for rescue. Rescue, in vivo, is likely to be dependent, initially, on interaction with antigen held on follicular dendritic cells (FDC). A subset of FDC located in that part of the germinal center furthest from centroblasts is particularly rich in CD23. Supernatants containing high levels of soluble CD23 were found not only to encourage the survival of germinal center B cells but also to promote their differentiation toward a plasmacytoid morphology; these activities were diminished following removal of CD23 from the supernatants. Recombinant 25-kDa CD23 was initially found to be incapable of providing the signal for germinal center cell development but on the addition of interleukin 1 alpha which, by itself, was inactive, rescue and differentiation of germinal center B cells were now achieved. Apoptosis in germinal center cells could also be prevented by the ligation of surface CD40 with monoclonal antibody: however, rescue via this pathway was not accompanied by plasmacytoid differentiation. These findings provide a functional rationale to the high level expression of CD23 found within a discrete subset of FDC and indicate a bifurcation in the development of germinal center B cells following their rescue from apoptosis.

Antigens, Differentiation, B-Lymphocyte↗

Germinal-center cell proliferation in response to T-independent antigens: a stathmokinetic, morphometric and immunohistochemical study in vivo.

Although the nature of the germinal center reaction during responses to T-dependent antigens has been well documented, much less is known regarding the relationship between germinal centers and T-independent antigens. In this study, germinal-center cell proliferation was determined at specific time points in spleens of C3H/HeN mice following immunization with either the type-1, T-independent antigen dinitrophenol-lipopolysaccharide (DNP-LPS), or the type-2, T-independent antigen DNP-Ficoll. A stathmokinetic technique was employed to assess proliferation in terms of germinal center cell birth rate and morphometry was used to measure actual growth and regression of the germinal center cell population. An estimate of the absolute rate of germinal-center (GC) cell proliferation was derived from these two values. In addition, immunohistochemistry was performed to correlate changes in GC cell proliferation with the presence or absence of antigen within GC. Following immunization with both antigens, there was an initial reduction of proliferation within pre-existing germinal centers which manifested as either GC dissociation (DNP-LPS) or a suppression of birth rates (DNP-Ficoll). This was followed by a period of increased GC cell proliferation in animals immunized with DNP-LPS, but not in those exposed to DNP-Ficoll. GC cell proliferation was then measured in mice treated with cyclosporin A from 1 day before to 2 days after immunization with DNP-LPS. In these animals, the expected increase in GC cell birth rates did not take place. Immunohistochemistry showed that DNP-Ficoll and DNP-LPS were present in GC from 1 day after immunization until the end of the experiment on day 7. Treatment with cyclosporin A did not affect the deposition of DNP-LPS in GC. These results show that only some T-independent antigens are able to stimulate GC cell proliferation, and we propose that this is related to their ability to recruit precursors of GC B cells into the GC reaction. In addition, the results indicate that GC proliferation seen in response to a so-called T-independent antigen is at least partly driven by T cell-derived cytokines.

Animals↗

Functional and electrophoretic characteristics of faba bean (Vicia faba) flour proteins as affected by germination.

Faba beans (Vicia faba) were germinated at room temperature for 3 and 6 days respectively. The effect of germination on the protein fractions, protein solubility index, PAGE pattern and some functional properties i.e. emulsification capacity (EC), foaming capacity (FC), foam stability (FS), water and fat absorption capacities of the flour was studied. Germination decreased albumins, globulins and prolamins at different levels but non protein nitrogen and glutelins were increased. The protein solubility index was high at both extreme pH values with an isoelectric point (IP) at pH of 4.4-4.5. The solubility of the protein slightly increased due to germination at all the pH values. PAGE pattern revealed on obvious dissociation and utilization for both fast and slow moving protein fractions during germination. Emulsification and foaming properties vs pH profile were similar to the pattern of solubility vs pH. Both properties were high at acidic and alkaline pH's and the minimum values were at pH 4 to 5. Germination process improved EC, FC and FS of the flour in comparison with that of dry bean flour. Water absorption of faba bean flours was improved during germination but the fat absorption markedly decreased.

Dietary Fats↗

Imaging and analysis of Bacillus anthracis spore germination.

External and internal changes occurring during the process of germination of Bacillus anthracis spores were observed through atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. AFM studies showed that in response to L-alanine (4 mM), as a germinant, the spore germinates into a vegetative cell in 3 hours. The temporal size changes occurring during the germination were gradual but the major change in size was observed between the second and third hour. TEM of spores showed the presence of varied layers, which is in accordance with previous studies. However, the integrity of these layers was lost gradually during the process of germination. The inner spore membrane remains intact even until late stages of germination, whereas the coat, outer spore membrane, and the cortical layers are discarded at the second-hour stage. The results indicate that sequential changes during the germination of a B. anthracis spore are similar to other species of the Bacillus group.

Bacillus anthracis↗

[Molecular biology of the germination of Bacillus spores].

The review deals with recent results and problems of gene expression during germination of Bacillus spores. Three problems were selected: 1. The activation of metabolism as a prerequisite for the synthesis of nucleic acids and proteins. 2. The activation of nucleic acid and protein synthesis during germination. 3. The gene expression programme of germinating spores. Using the highly sensitive two-dimensional polyacrylamide gel analysis three major classes of proteins were distinguished, depending on the time of onset and duration of their syntheses: a) proteins made throughout germination (main class), b) proteins whose synthesis started only after a lag phase and then continued throughout germination, and c) proteins which are synthesized only during the early phases of germination. The programme of protein synthesis is an indicator for the control of gene expression during germination. The regulation of expression of these major gene groups during spore outgrowth is discussed.

Bacillus↗

Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores.

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.

Bacillus anthracis↗

The effect of humidity on germination and infection of termites by the hyphomycete, Metarhizium anisopliae.

The effect of relative humidities (r.h.) from 90 to 100% on germination of a termite-active isolate of Metarhizium anisopliae (isolate FI25 and FI610) was studied using a liquid germinating medium to which the appropriate amount of glycerol had been added. Germination was increasingly delayed at water activities equivalent to 99, 98, and 96% r.h. and completely inhibited at 94, 92, and 90%. Twenty-one isolates were then screened for germination at 96 and 100% r.h. All isolates showed delayed germination at 96% r.h. but most isolates eventually gave a high final percentage germination at this humidity. Two isolates, FI527 and FI638, were markedly slower to germinate at both humidities. The susceptibility of two species of termites, Nasutitermes exitiosus and Coptotermes acinaciformis, to FI610 was tested at r.h. down to 86%-the lowest humidity at which the insects would survive. No consistent effect of humidity on pathogenicity was detected. Mortality occurred over the range range of humidities tested; sporulation from the disease-killed termites, however, occurred only at r.h. above 93%. It is concluded that the microclimate around living termites is usually sufficiently humid to ensure infection under most field conditions and that humidity is unlikely to limit the efficacy of the fungus in controlling termites.

Animals↗

Nutrient requirements in germination of conidiospores of Aspergillus niger V. Tieghen.

Germination of conidiospores of A. niger was stimulated by the addition of some carbohydrates and nitrogenous compounds to the growth medium. Glucose at 0.5 and 1.0 mg/ml concentrations was the best sugar for germination. Sucrose and fructose were the second best. Glutamic acid and valine were the most effective nitrogenous compounds for the spore germination of the fungus. Glucose, sucrose, and glutamic acid initiated germination within the first 6 h of incubation. When cellobiose, galactose and valine were used germination started after 9 h. With leucine, cysteine and arginine, however, not only the percentage of germination reduced, but also the latent period increased, significantly. A period of 15 h was needed before germination had been started.

Amino Acids↗

Volatile and gaseous metabolites released by germinating seeds of lentil and maize cultivars with different susceptibilities to fusariosis and smut.

The effect of volatile and gaseous metabolites released by germinating seeds of lentil cultivars more and less susceptible to fusariosis on the germination of spores of Mucor racemosus, Trichoderma viride, Verticillium dahliae and Botrytis cinerea was found to depend rather on the fungal genus than on the lentil cultivar. However, spores of Fusarium oxysporum reacted more sensitively during germination to the presence of exudates of both cultivars, when the more susceptible lentil displayed a stimulation, the less susceptible one an inhibition of spore germination. The greatest difference in the effect of exudates was observed in the more and less susceptible maize cultivars with respect to the germination of chlamydospores of Ustilago maydis, especially during the first hours of seed germination. Analysis of the exudates of germinating seeds showed the release of a greater amount of ethanol and methanol with acetaldehyde by the more susceptible cultivars of lentil and particularly maize.

Ethanol↗

A method for extracting RNA from dormant and germinating Bacillus subtilis strain 168 endospores.

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid-phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10(8) spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.

Bacillus subtilis↗

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores.

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.

Cellulase↗