Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

Effects of zinc on macromolecular synthesis and nuclear division during the yeast to mycelium transition in Sporothrix schenckii

Zinc ions (10 mM) have been reported previously to inhibit the yeast to mycelium transition in Sporothrix schenckii. Yeast cells of this fungus were harvested, selected by filtration and allowed to form germ tubes in a basal medium with glucose in the presence of 10 mM zinc and the effects of this ion on protein, RNA and DNA synthesis and nuclear division recorded. All of these processes were affected by the addition of 10 mM zinc to the medium. Nevertheless, the inhibition of protein synthesis was observed earlier than that of RNA or DNA synthesis and was of a greater magnitude than that observed for both of these processes. Protein synthesis was inhibited within the first hour after inoculation, at which time this process begins in the control cells. RNA synthesis was inhibited during the 3 to 6 h interval after inoculation, that is, 3 h after the start of this process in the control cells. After 9 h of incubation, the inhibition of protein synthesis had reached its maximum at 70%, while that of RNA synthesis was only 52%. DNA synthesis was slightly inhibited, with maximum inhibition being observed 9 h after inoculation. Nuclear division in cells forming germ tubes in the presence of 10 mM zinc took place with a 3 h delay in relation to the control cells. These observations suggest that the inhibition of protein synthesis might be the most important mechanism by which zinc inhibits the yeast to mycelium transition in S. schenckii.     

Ethanol-induced germ tube formation in Candida albicans

Ethanol is the first reported compound which can induce germ tube formation in Candida albicans without the addition of any nitrogen-containing nutrients. Conditions controlling induction of germ tubes in C. albicans by ethanol were investigated. Ethanol (17.1 mM) in buffered salts solution containing sodium bicarbonate induced 70 to 80% of yeast phase cells of C. albicans to form germ tubes. Germ tubes could be induced by ethanol (0.08 to 340 mM) at temperatures ranging from 29 to 41 degrees C (optimum 37 degrees C) and at pH values ranging from 3.0 to 8.0 (optimum 5.75). The germ tubes averaged 11 micron in length after 6 h at 37 degrees C. The percentage of cells forming germ tubes decreased as the concentration of cells in the induction solution was increased above 4 X 10(5) cells ml-1. Germ tubes first appeared 45 to 60 min after continuous exposure to ethanol at 37 degrees C and all cells which formed germ tubes did so by 2 h. Germ tube length decreased as the pH was increased but was independent of the concentration of ethanol. Oxygen was required for germ tube formation. In addition to ethanol, 1-propanol, 2-propanol, 1-butanol and acetic acid could induce germ tube formation, whereas methanol could not. These results indicate that the cells must mobilize their endogenous nitrogen and probably carbohydrate reserves in order to initiate formation of germ tubes. The evidence is inconclusive as to whether ethanol itself must be metabolized for germ tube induction to occur, although it is not thought to act by a nonspecific interaction with the cell membrane.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

The requirements for bicarbonate and metabolism of the inducer during germ tube formation by Candida albicans

Factors affecting germ tube formation in Candida albicans at suboptimal temperatures were investigated. Candida albicans formed germ tubes between 22 and 30 degrees C in solution when incubated without shaking, in the presence of bicarbonate (2 mg mL-1). Other conditions depended on the inducer used. Proline could induce germ tube formation optimally only when its concentration was between 200 and 400 mM. A concentration of 0.05 mM N-acetylglucosamine was sufficient to induce germ tube formation. N-Acetylglucosamine could induce germ tube formation at 30 but not at 25 degrees C. N-Acetylglucosamine induced germ tube formation was most reproducible when the cells were first starved by incubation in water for 16-24 h at 20 degrees C. Germ tubes induced by proline could be formed at pH values between 3.8 and 9.0 at 30 degrees C, but only between 7.0 and 7.5 at 25 degrees C. The addition of 0.05 to 5 mM glucose to a 5 mM proline induction solution allowed germ tube formation at 30 but not at 25 degrees C. Glucose (400 mM) did not suppress germ tube formation at 30 degrees C but only 5 mM was sufficient to cause a 65% suppression at 25 degrees C. The results show the importance of CO2 and (or) bicarbonate to the induction of germ tube formation and are consistent with the metabolism of the inducer.     

Overexpression of the actin gene is associated with the morphogenesis of Candida albicans

A progressive increase in the synthesis of actin mRNA was observed by Northern blot analysis, when cells were induced to form germ tubes at 37 degrees C by N-acetyl-D-glucosamine. Presence of trifluoperazine, a calmodulin inhibitor, or incubation of cells at 25 degrees C, or by replacing N-acetyl-D-glucosamine with glucose which inhibited germ tube formation lowered this synthesis. Furthermore, in vitro translation of total RNA revealed an increase in the synthesis of actin (45 kDa) during germ tube formation. These results suggest for the first time that the expression of actin gene is regulated during morphogenesis of C. albicans.     

Early development in an algal gametophyte: role of the cytoskeleton in germination and nuclear translocation

Summary Biflagellate zoospores from the giant kelpMacrocystis pyrifera underwent germination after adhering to a substrate and produced germ tubes that were approximately 13–15 m in length. Coincident with the germ tube elongation was organelle (other than the nucleus) translocation along the tube. Shortly after formation of the germ tube, the zoospore nucleus divided and one daughter nucleus translocated along the germ tube. The nucleus did not appear to undergo chromosomal condensation prior to division. The nuclear division and/or translocation of the daughter nucleus did not begin until well after germ tube elongation was complete, demonstrating that these are temporally distinct developmental events. The translocation of one daughter nucleus coincided with differentiation of the distal end of the germ tube into a bulbous structure. Following this, the first gametophytic cross wall was formed and, subsequently, the daughter nucleus remaining in the original zoospore body underwent repositioning, assuming a position in the germ tube near the cross wall. Cytochalasin D inhibited germ tube elongation suggesting that actin microfilaments are probably involved in this developmental process. In addition, when cytochalasin D was added to the culture after the germ tube elongation was complete, it did not affect either nuclear division or translocation, indicating that microfilaments were not directly involved in these nuclear events. Colchicine and the plant specific microtubule disrupting agent, amiprophos methyl blocked nuclear division and translocation without affecting germination or germ tube elongation. These data suggest that actin microfilaments are primarily responsible for complete germination, specifically germ tube elongation, while microtubules are involved in nuclear division and translocation. The present study demonstrates that germination (and germ tube elongation) and nuclear translocation inM. pyrifera gametophytes are temporally and mechanistically distinct developmental events.     

Growth and cell division of Mycobacterium avium

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.     

Differential deoxyribonucleic acid synthesis during microcycle conidiation in Neurospora crassa

In conidia of Neurospora crassa germinating at 25°C, DNA synthesis measured by incorporation of tritiated adenosine reaches a maximum soon after the outgrowth of the germ tube (6–7h after inoculation). In conidia heat-treated at 46°C (for 15h), a maximum of incorporation of the DNA precursor occurs already 1h after inoculation, then the incorporation progressively declines until the end of the heat-shock. When such conidia are shifted to 25°C, a maximum of DNA synthesis occurs during the development of the presumptive conidiophore as at the outgrowth of normal germ tubes. This wave of DNA synthesis is followed by a second maximum of DNA synthesis, occurring only in the microcyclized cultures, when the premature differentiation of proconidia takes place. Prevention of this second wave of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine respectively reduces or fully inhibits such induced conidial differentiation.     

Appressorium formation and nuclear division in Colletotrichum truncatum

Conidia of the soybean anthracnose fungus, Colletotrichum truncatum differentiate to form appressoria required for host invasion when the germ tube touches a hard surface. This thigmotrophic stimulus appears to be translated by the fungus during the second round of nuclear division. Inhibiting the second round of DNA synthesis by fluorodeoxyuridine or hydroxyurea blocked appearance of appressoria but not emergence of the germ tube. DNA synthesis and mitosis resumed upon removal of FUdR but only mycelia formed, and infection structures did not appear. In addition, actinomycin D reversibly blocked development of appressoria and synthesis of polyadenylate, but nuclear division was not affected. The data suggest that anthracnose conidia produce appressoria in response to germ tube contact by altering the messenger program of its germ tube nucleus. This study has also shown that mitochondrial DNA had an unusual bimodal distribution in CsCl at 1.690 and 1.719 g/cm³, respectively.Non-Standard Abbreviations FUdR 5-fluorodeoxyuridine - polyA polyadenylic acid     

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.     

Cellular events in growth and germination of giant conidia of Neurospora crassa

During growth of conidia in 3.22 M ethylene glycol the increase in the number of the nuclei is proportional to the increase in volume only in the phase of maximum growth rate and is lower in the preceding and in the following periods of growth. DNA synthesis similarly initiates later and decreases faster than protein synthesis. The dilution of ethylene glycol is followed by the germination of giant conidia, which is characterized by the absence of a lag phase, a high degree of synchrony and the formation of more than one germ tube per conidium. The number of germ tubes is dependent on the volume reached by conidia at the end of the treatment and does not increase with time. The resuming of DNA synthesis after germination is preceded by a sharp increase in protein synthesis and the division of almost half of the nuclei and shows a synchronized pattern. Results are discussed in the light of models of growth proposed for eukaryotic cells.     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

Physiology of spore germination in cephalosporium acremonium

Conidia ofC. acremonium require an exogenous supply of carbon, nitrogen, magnesium, and phosphate for swelling and germ tube formation. Germination is stimulated by supplementing the medium with sulfate. Maximum frequency of germination occurs at a temperature of 27° to 32°C and a pH of 8.0. Conidia swell at pH 4.0 to 5.5 but do not form germ tubes. Conidia allowed to swell at pH 5.5 initiate germ tube formation immediately when the pH is adjusted to 7.5. Under optimal conditions, over 95 percent of the spore population formed germ tubes by 13 hours.     

Synchronous Cell Division in Cultured Explants of Jerusalem Artichoke Tubers: the Effects of 5-Fluorouracil on Messenger RNA Synthesis and the Induction of Cell Division

Explants of secondary xylern parenchyma tissue from Jerusalemartichoke tubers were induced to undergo cell division and de-differentiateby culture in nutrient medium. The first division was inherentlysynchronous. The system was used to study the involvement ofmessenger RNA synthesis in the induction and continuance ofcell division in previously non-dividing cells. The base analogue 5-fluorouracil (5-FU) inhibited ribosomalRNA synthesis and the processing of ribosomal RNA precursorto mature 25 S and 18 S RNAs. The synthesis of messenger-likeRNAs (heterogeneous in size, labelled to a high specific activityin a pulse incubation, and containing a polyadenylic acid sequence)was less inhibited by 5-FU. Explants grown in 5-FU did not synthesize DNA and did not divide.A direct inhibition of DNA synthesis by 5-FU added late in culturewas reversed by thymidine. An indirect inhibition of DNA synthesisoccurred when 5-FU was present from the start of culture andwas not reversed by thymidine. Because ribosomal RNA synthesisis not necessary for the induction of cell division (Fraser,1975) and because 5-FU was incorporated into mENA, probablyinterfering with its function, these results suggest that 5-FUinhibited the metabolism of mRNA which was required for DNAsynthesis and cell division. The timing of mRNA synthesis required for DNA synthesis andcell division was investigated by adding 5-FU plus thymidineto cultures at various times. By the beginning of DNA synthesisfor the first division, explants were competent, in terms ofmRNA synthesized, to complete the first division. MessengerRNA synthesis occurring before the end of the first divisionallowed explants to undergo at least three more divisions.     

Cytological interrelationships between the cell cycle and duplication cycle of Candida albicans

The cytology of nuclear division and septation in the yeast and hyphal phases of Candida albicans growing at 37 degrees C has been studied by fluorescence microscopy after staining of specimens with 4'6-diaminido-2-phenylindole (DAPI) and Calcofluor. Yeast and hyphal cells replicated their nuclei at about 18 min after the emergence of a bud or germ-tube. The site of nuclear division coincided with the future location of the septum in both forms. This occurred at the junction of the bud and parent yeast cell or 6.0 micron from the parent yeast in germ tubes which were formed in medium containing serum. The filamentous forms of a range of clinical and laboratory strains grown in a variety of germ tube-inducing media were all extensively vacuolated. Germ tube extension in all of these media was linear. It is suggested that there is little biosynthesis of cytoplasm during the initial stages of germ tube growth in this organism and that this accounts for the development of the large vacuoles and the linear growth kinetics.     

Deoxyribonucleic acid metabolism and nuclear division during spore germination in Fusarium oxysporum.

Ungerminated microconidia of Fusarium oxysporum have a mean cell DNA content of 0-134 times 10--12 g/cell with a guanine-plus-cytosine composition (%GC) of 50-75%. During germination, the first dry weight increase of the spore population was defected after 3 h incubation and the first germ tube appeared after 4 h. The total DNA of the culture sharply increased after 5 h, followed by a pause at 6 h. At this time the DNA content per nucleus was maximal and the first nuclear divisions were detected. auses in the rise of total DNA of the culture and in the [14C]adenine incorporation pattern suggest that there is partial synchrony in DNA synthesis at the beginning of incubation. This is also supported by the fact that until 8 h, only hyphae with 1, 2 and 4 nucleic were observed. [14C]adenine incorporation into DNA averaged 2-68% of the total taken up in 10 h incubation.     

Cytoplasmic alkalinization during germ tube formation in Candida albicans

Weak acids were used to measure the internal pH of yeast cells of Candida albicans that had been induced to form buds or germ tubes. Under conditions that supported germ tube formation the internal pH rose from around 6.8 to over 8.0 after 30 min in two different induction media. Internal pH measured by 31P NMR confirmed this pattern and also showed that the internal pH fell to around 7.0 prior to the outgrowth of germ tubes. Conditions which led to budding induced less cytoplasmic alkalinization. This alkalinization was brought about when cells were inoculated into media of neutral pH and at an increased temperature. Increasing the temperature of the medium augmented the alkalinization of the cytoplasm induced by raising the external pH. Strains of C. albicans defective in the ability to produce germ tubes did not show this dramatic cytoplasmic alkalinization under conditions which normally supported filamentous growth. The raising of internal pH may be due to the activation of the plasma membrane proton-pumping ATPase since diethylstilboestrol inhibited the cytoplasmic alkalinization and germ tube formation without causing irreversible loss of cell viability. The results show that the induction of the dimorphic transition in this organism is accompanied by a steep rise in internal pH. It is not known whether these changes are the cause or consequence of morphogenesis.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.