Developmental stage-dependent response ofPilobolus crystallinus sporangiophores to gravitative and centrifugal stimulation

Gravitropic response of sporangiophores ofPilobolus crystallinus was studied by successive microscopic observation of the sporangiophores horizontally placed in the dark (red light) and by analysis of sporangiophore response to centrifugal stimulation. Negative tropism against the gravitative and also centrifugal stimulation was found only in mature sporangiophores after development of sporangium and after the resumption of elongation beneath the fully-developed subsporangial vescle, but there was no response in younger sporangiophores, implying that the gravitative perception system of the sporangiophores is dependent on their developmental stages.     

MEMBRANE POTENTIAL AND ENDOGENOUS ION CURRENT OF PHYCOMYCES SPORANGIOPHORES

Glass microelectrodes were inserted into the growing zone of sporangiophores of Phycomyces blakesleeanus that had been submersed in artificial pond water. The membrane potential (inside negative) increased with increasing pH of the bathing solution from an average of ?98 mV at pH 5 up to ?131 mV at pH 7. Removal of Ca²⁺ from the medium hyperpolarized the membrane potential in the wild type, but caused a significant depolarization in the blue-light-insensitive madC mutant. KCN, diethylstilbestrol, and N,N′-dicyclohexylcarbodiimide depolarized the membrane potential in both the wild type and the madC mutant, while fusicoccin had no effect. Endogenous ion current of up to 2 μA cm^?2 was measured in the growing zone of sporangiophores with an extracellular vibrating electrode. The current density and current pattern varied with the pH of the medium. At pH 5 most sporangiophores had weak inward current along the growing zone, whereas at pH 7 most sporangiophores had strong outward current. The response of the membrane potential to specific inhibitors and the presence of an endogenous ion current indicate an electrogenic H⁺-ATPase in the plasma membrane. The results show a negative correlation between growth rate of sporangiophores growing in buffered aqueous medium and magnitude of membrane potential, as well as density of outward current. They also indicate an important role of protons in controlling the growth of Phycomyces sporangiophores.     

Mutants ofPhycomyceswith Decreased Gallic Acid Content

Weinkove, D., Poyatos, J. A., Greiner, H., Oltra, E., Avalos, J., Fukshansky, L., Barrero, A. F., and Cerdá-Olmedo, E. 1998. Mutants ofPhycomyceswith decreased gallic acid content.Fungal Genetics and Biology25, 196–203. Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycetePhycomyces blakesleeanus,much more so than in the basal mycelium or the culture medium. The actual concentrations vary with illumination, age of the culture, and composition of the medium. We devised a simple screening procedure to isolatehbamutants whose sporangiophores contained less gallic acid than the wild type. The most useful mutant had very low concentrations of hydroxybenzoic acids in the sporangiophores, but about the same as the wild type in the basal mycelium and the medium. The mutant was only slightly different from the wild type in growth and morphology. Mutant and wild-type sporangiophores grew away from ultraviolet C sources (260 nm) equally well. Contrary to previous conjectures, ultraviolet tropism does not depend on the ultraviolet absorption of gallic acid or other free hydroxybenzoic acids in the sporangiophore. Against expectations, phenols did not impair DNA extraction: sporangiophores produced better DNA preparations than basal mycelia and thehbamutant only slightly better than the wild type.     

Morphogenesis of leaves and cones of male short-shoots of Ginkgo biloba L.

Although the subject of several studies, the phylogeny of Ginkgo biloba is still ambiguous. Most of the morphological and some palaeontological studies assume a close relationship to conifers, but other palaeontological studies regard the origin of Ginkgo biloba in groups that exhibit a pinnate bauplan like Peltaspermales or Dicranophyllales. This divergence has led to controversial interpretations of male sporangiophores and leaves. Attempting to resolve this, here we have investigated the male cones and leaves of short-shoots by SEM and light-microscopy. Our results indicate that the male sporangiophores are simple structures, and the observed formation of thickened cell walls at the sterile adaxial side of the sporangiophores, similar to the endothecium of the sporangia, gives weak support for a precursor of Ginkgo-sporangiophores that displayed simple male sporangiophores with radial arrangement of the sporangia. Thus, our interpretations of the male sporangiophores of Ginkgo biloba allude to a relationship with Coniferales, Gnetales and Cordaitales and reject a close relationship of Ginkgo biloba with pinnate groups like Cycadaceae, as assumed by some molecular studies. In contrast to previous studies on long-shoot leaves, our results on short-shoot leaves give no indication for a compound character of Ginkgo leaves. Moreover, we infer that Ginkgo leaves could be derived from a simple bauplan, by two modifications of the basic growth pattern of conifer leaves, assuming that the dissection of Ginkgo leaves is secondary. Although more comparative investigations are necessary, our results support a coniferophyte origin of the Ginkgoales.     

An efficient grafting system for transgenic plant recovery in cotton (Gossypium hirsutum L.)

A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacterium infection and kanamycin selection was developed. Various aspects of in vitro grafting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitro grafting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitro seedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitro grafting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors.     

Diameter and amount of β-carotene determine the maximal phototropic bending angle ofPhycomyces sporangiophores

Wild-type sporangiophores at stage IVb (final developmental stage after sporangium formation) ofPhycomyces show a pronounced positive phototropism to unilateral white light. We found that the maximal bending angle was larger in thin sporangiophores than in thick ones, and larger in the sporangiophores containing a small amount of β-carotene than in those containing a large amount of it. These phenomena probably occur because of the increase in length of intracellular light path or in the intracellular light-attenuation coefficient, as supported theoretically.     

Gravitropism in Phycomyces: a role for sedimenting protein crystals and floating lipid globules

To elucidate the graviperception of the unicellular fungus, Phycomycesblakesleeanus, sporangiophores were inspected for intracellular structures which relocate with respect to gravity. Two structures, paracrystalline proteins (so-called octahedral crystals) and an aggregate of lipid globules, were identified which showed redistribution upon reorientation of the sporangiophore. Octahedral crystals occur throughout the sporangiophore, including the apical growing zone, and are localized inside vacuoles in which they reside singly or in clusters of up to 40 loosely associated individuals. Upon a 90° reorientation of sporangiophores, crystal clusters sedimented in approximately 50–200 s from the upper to the lower side, corresponding to a speed of 0.5–2 μm s⁻¹. Stage-4 sporangiophores (with sporangium) of three mutants which lack the crystals displayed anormal kinetics of gravitropism and substantially reduced bending angles in comparison to sporangiophores of the wild type. While horizontally placed wild-type sporangiophores reached the vertical position after 10–12 h, the crystal-lacking mutants bent maximally 40°–50° upward. In stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 μm below the apex. The globules floated upwards when the sporangiophore was placed horizontally forming in this way a cap-like aggregate. It is proposed that both the sedimenting protein crystals and the upward-floating globules are involved in gravisensing. Received: 23 March 1999 / Accepted: 27 May 1999     

Blue-light control of sporangiophore initiation in Phycomyces

Summary Many fungi produce spores or spore-bearing structures under the control of blue light. Sporangiophores of Phycomyces blakesleeanus are produced continuously along racing tube cultures grown in constant darkness or constant light. However, if a dark-grown culture is exposed to light for a short time on one day a narrow, dense band of sporangiophores is observed the next day at that point of the tube occupied by the mycelial tips during the light pulse. A periodic program with short days (e.g., 4 h light; 20 h dark), leads to periodic bands of sporangiophores spaced at intervals corresponding to one period-length (in this case 24 h) of mycelial growth. Sporangiophore initiation is inhibited by a light to dark transition and is stimulated by a dark to light transition. A partial action spectrum of the initiation response, covering the critical 480–540 nm region, strongly suggests that the same photoreceptor pigment is involved as in the phototropic response and light growth response of sporangiophores. Mutants with altered light control of sporangiophore initiation have been found among those selected for altered phototropism. This joint elimination of these two responses to blue light by a single mutation is evidence for a common early transduction system.     

Positional differentiation for development along the sporangiophore in <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

 Small segments of Phycomyces sporangiophores regenerate various structures on incubation in a moist chamber. We tested the regeneration capacities of middle sporangiophore segments whose protoplasm had been totally or partially removed and replaced with protoplasm from various segments of a genetically different strain. The structures that were regenerated depended on the source of the injected protoplasm (sporangia of various sizes and segments from middle and basal parts of the sporangiophores), implying a positional differentiation of protoplasm along the sporangiophore axis. Protoplasm from various sources showed a high affinity; that is, they mixed successfully and led, in most cases, to the formation of heterokaryotic regenerating structures. The highest affinity was seen when mixing protoplasm from the middle segments of two different strains. Received: November 29, 2000 / Accepted: March 19, 2002     

Growth-rate adaptation of Phycomyces sporangiophores to partial depletion of oxygen

The growth rate of Phycomyces blakesleeanus sporangiophores was found to be very sensitive to sudden changes in the oxygen concentration. A change from 20% to 15% oxygen elicits a transient decrease in the growth rate which returns to normal 10 min after altering the concentration. After a step change to 10% oxygen, the growth rate shows two minima at 6–8 and 30–35 min and it reaches about 80% of its original value 50 min after this change. A threshold curve for this negative growth response shows that sporangiophores begin to sense a decrease in the oxygen concentration from 20% to 17%. Seven phototropically abnormal mutants with defects in the genes madA to madG were tested for the oxygen response. Two strains, C149madD120 and C316madF48, were found to have recoveries different from those of the wild type after step changes from 20% to 10% oxygen.     

Morphogenesis of the reproductive shoots of Welwitschia mirabilis and Ephedra distachya (Gnetales), and its evolutionary implications

For decades, Gnetales appeared to be closely related to angiosperms, the two groups together forming the anthophyte clade. At present, molecular studies negate such a relationship and give strong support for a systematic position of Gnetales within or near conifers. However, previous interpretations of the male sporangiophores of Gnetales as pinnate with terminal synangia conflict with a close relationship between Gnetales and conifers. Therefore, we investigated the morphogenesis of the male reproductive structures of Welwitschia mirabilis and Ephedra distachya by SEM and light microscopy. The occurrence of reduced apices to both halves of the antherophores of W. mirabilis gives strong support for the assumption that the male ‘flowers’ of W. mirabilis represent reduced compound cones. We assume that each half of the antherophore represents a lateral male cone that has lost its subtending bract. Although both halves of the antherophores of Ephedra distachya lack apical meristems, the histological pattern of the developing antherophores supports interpreting them as reduced lateral male cones as well. Therefore, the male sporangiophores of Gnetales represent simple organs with terminal synangia. Although extant conifers do not exhibit terminal synangia, similar sporangiophores are reported for some Cordaitales, the hypothetical sister group of conifers. Moreover, several Paleozoic conifers exhibit male cones with terminal sporangia or synangia. Therefore, we propose that conifers, Cordaitales and Gnetales originated from a common ancestor that displayed simple sporangiophores with a terminal cluster of sporangia.     

Segregation of heterokaryons in the asexual cycle of Phycomyces

Summary Artificial heterokaryons between carotene mutants ofPhycomyces blakesleeanus Bgff. have been prepared by squeezing cytoplasm out of two different mutant sporangiophores and allowing the fused droplets to regenerate.These heterokaryons are used to study the distribution of nuclei at different stages of the asexual life cycle. It is proposed that the nuclear ratio is constant in all parts of the mycelium, sporangiophores and sporangia, and that random samples of nuclei are packaged into spores. This model permits quantitative predictions regarding the proportions of phenotypes in the asexual progeny and these predictions are corroborated by experiments. The nuclear ratio remains constant during repeated mycelial transfers.     

A NEW BOWMANITEAN FRUCTIFICATION FROM THE PENNSYLVANIAN OF KANSAS

Mamay , Sergius H. (U. S. Geological Survey, Washington, D. C.) A new bowmanitean fructification from the Pennsylvania of Kansas. Amer. Jour. Bot. 46(7): 530–536. Illus. 1959.—A new sphenophyllaceous fructification, Bowmanites moorei sp. nov., is described on the basis of coal ball petrifactions from the Fleming coal in southeastern Kansas. One of the simplest species of Bowmanites, B. moorei has only 3 sporophylls in a whorl. Each sporophyll has 3 lobes, the median one fertile, the 2 lateral ones sterile. Each fertile lobe subtends 2 sporangia, resulting in 6 sporangia in a whorl. The sporangia are inverted, being distally attached to separate sporangiophores that arise in pairs, in axillary positions. The sporangial epidermis and epidermis of the lower surfaces of the sporangiophores are characterized by relatively large, thick-walled cells. The spores are small, spherical, and trilete, without distinctive ornamentation of the exine. B. moorei adds to the morphological variation known in Bowmanites, but contributes little toward a phylogenetic interpretation of this morphologically complex genus.     

Complementation betweenPhycomyces mutants of mating type (+) with abnormal phototropism

Summary Twelve mutants ofPhycomyces blakesleeanus with defects in sporangiophore phototropism (genotypemad) were obtained from a wild type of the (+) mating type by mutagenesis with nitrosoguanidine. These mutants were tested for genetic complementation against standard (+)mad mutants derived from sexual crosses between the isogenic (+) strain and established (-)mad mutants (Ootaki et al., 1974; Eslava et al., 1976). Heterokaryons for complementation tests were obtained by grafting stage I sporangiophores. The (+) mutants were also investigated for their sensory responses such as photoinduction of sporangiophores and avoidance. The mutants were grouped into two classes, based on the phenotypic classification scheme of Bergman et al. (1973). There were eleven class 1.2 mutants and one class 2 mutant. Complementation tests revealed that all eleven class 1.2 mutants carry the genemadC and the class 2 mutant carriesmadD. There was no evidence that any were double mutants. These results are consistent with the phenotypic classification and with the complementation results of themad mutants of the (-) mating type.     

The Effects of Calcium and the Ionophore A23I87 on Modulation, Nitrogen Fixation and Growth of Soybeans

Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five-day-old cultures of Phycomyces blakesleeanus show notice-able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium. The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.     

Oxygen consumption of Phycomyces sporangiophores

Summary The respiration of stage-I,-III, and-IV sporangiophores of Phycomyces was measured. The of stages-I and-IV is low compared to that of actively growing mycelium. The of stage-III sporangiophores is somewhat higher than that of stages-I and-IV. As stage-I sporangiophores elongate their respiration rate increases proportionately. The respiration rate of stage-IV sporangiophores does not change as they elongate. In stage-IV sporangiophores the growing zone and the columella together consume as much oxygen as the rest of the sporangiophore does. Changes in the sporangiophore's growth rate have no effect on its oxygen consumption.This work was submitted in partial fulfillment of the requirements for the Ph. D. degree.     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism

 The negative gravitropism of the sporangiophores of Phycomyces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132–142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15–25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains. Received: 2 September 1999 / Accepted: 9 November 1999     

Bone grafts as vancomycin carriers in local therapy of resistant infections

The level of an antibiotic capable of inhibiting the etiological agent at the site of infection is an essential prerequisite for successful antibiotic therapy. In some cases, locally applied antibiotics may compensate for limitations of systemic administration and shorten systemic therapy. We aimed at verifying to what extent vancomycin (Van) bound to ground bone grafts is usable in the treatment of serious infections. The levels of released Van significantly exceeded the Van minimum inhibitory concentration, which can suppress Van-sensitive staphylococci and Van intermediate Staphylococcus aureus, for the whole period of a 16-day measurement. Our results indicate that bone grafts can be used as Van carriers in therapy of osteomyelitis caused by Van-sensitive Staphylococcus strains.     

A comparison of cell-wall-yielding properties for two developmental stages of Phycomyces sporangiophores

The yielding properties of the cell wall, irreversible wall extensibility (m) and yield threshold (Y), are determined for stage I sporangiophores of Phycomyces blakesleeanus from in-vivo creep experiments, and compared to the values of m and Y previously determined for stage IVb sporangiophores using the same pressureprobe method (Ortega et al., 1989, Biophys. J. 56, 465). In either stage the sporangiophore enlarges (grows) predominately in length, in a specific region termed the growing zone, but the growth rates of stage I (5–20 urn · min^–1) are smaller than those of stage IVb (30–70 m · min^–1). The results demonstrate that this difference in growth rate is the consequence of a smaller magnitude of m for stage I sporangiophores; the obtained values of P (turgor pressure), Y, and P-Y (effective turgor for irreversible wall extension) for stage I sporangiophores are slightly larger than those of stage IVb sporangiophores. Also, it is shown that the magnitude of m for the stage I sporangiophore is regulated by altering the length of the growing zone, L_g. A relationship between m and L_g is obtained which can account for the difference between values of m determined for stage I and stage IVb sporangiophores. Finally, it is shown that similar changes in the magnitude of m and (which have been used interchangeably in the literature as a measure of irreversible wall extensibility) may not always represent the same changes in the cell-wall properties.Abbreviations and Symbols L length - L_g length of growing zone - m irreversible wall extensibility - P turgor pressure - Y yield threshold - (P-Y) effective turgor for irreversible wall extension - relative irreversible wall extensibility - _g relative irreversible wall extensibility of the growing zone (m/L_g) This work was supported by National Science Foundation grant DCB-8801717 to J.K.E. Ortega.