Phycomyces: An Increase in Mechanical Extensibility during the Avoidance Growth Response

The sporangiophore of Phycomyces shows a transient response to a double barrier, the avoidance growth response. Tensile tests conducted on the stage IV sporangiophore demonstrate that an increase in mechanical extensibility occurs about a minute after a double barrier stimulus. This change in mechanical extensibility is similar to the one that occurs after a light stimulus. We have concluded that the avoidance stimulus occurs somewhere on the same pathway between the photoreceptor mechanism and the final growth response.     

The Light Growth Response of Phycomyces

With the help of an automated tracking system we have studied the characteristics of the transient light growth response of Phycomyces. The response shows a sharply defined latency. The Q₁₀ of the reciprocal latency is 2.4. Response patterns at different peaks of the action spectrum are the same. The gradual variation of response magnitude over a wide range of adapted intensifies parallels that of phototropism. The responses to saturating stimuli exhibit a strong oscillation with a constant period of 1.6 min and variable damping. The growth responses to sinusoidally varying light intensities show a system bandwidth of 2.5 x 10^-3 Hz. The linear dependence of phase shift on frequency is largely attributable to the latency observed with pulse stimuli. In the high intensity range a previously suspected increase of the steady-state growth rate with intensity has been confirmed. The light growth responses of mutants selected for diminished phototropism have been investigated. Many of these mutants have sizable but grossly distorted growth responses.     

Avoidance of Phycomyces in a controlled environment.

The sporangiophore of the fungus Phycomyces bends away from nearby objects without ever touching them. It has been thought that these objects act as aerodynamic obstacles that damp random winds, thereby generating asymmetric distributions of a growth-promoting gas emitted by the growth zone. In the interest of testing this hypothesis, we studied avoidance in an environmental chamber in which convection was suppressed by a shallow thermal gradient. We also controlled pressure, temperature, and relative humidity of the air, electrostatic charge, and ambient light. A protocol was established that yielded avoidance rates constant from sporangiophore to sporangiophore to within +/- 10%. We found that avoidance occurred at normal rates in the complete absence of random winds. The rates were smaller at 100% than at lower values of relative humidity, but not by much. Remarkably, at a distance as great as 0.5 mm, avoidance from a 30-micron diam glass fiber (aligned parallel to the sporangiophore) was about the same as that from a planar glass sheet. However, the rate for the fiber fell more rapidly with distance. The rate for the sheet remained nearly constant out to approximately 4 mm. We conclude that avoidance depends either on adsorption by the barrier of a growth-inhibiting substance or emission by the barrier of a growth-promoting substance; it cannot occur by passive reflection. Models that can explain these effects are analyzed in the Appendix.     

Avoidance response, house response, and wind responses of the sporangiophore of Phycomyces

Avoidance response: An object placed 1 mm from the growing zone of a Phycomyces sporangiophore elicits a tropic response away from the object. The dependence of this response on the size of the object and its distance from the specimen is described, as well as measurements which exclude electric fields, electromagnetic radiation, temperature, and humidity as avoidance-mediating signals. This response is independent of the composition and surface properties of the object and of ambient light. House Response: A house of 0.5- to 10-cm diameter put over a sporangiophore elicits a transient growth response. Avoidance responses inside closed houses are slightly smaller than those in the open. Wind responses: A transverse wind elicits a tropic response into the wind, increasing with wind speed. A longitudinal wind, up or down, elicits a transient negative growth response to a step-up in wind speed, and vice versa. It is proposed that all of the effects listed involve wind sensing. This proposal is supported by measurements of aerodynamic effects of barriers and houses on random winds. The wind sensing is discussed in terms of the hypothesis that a gas is emitted by the growing zone (not water or any normal constituent of air), the concentration of which is modified by the winds and monitored by a chemical sensor. This model puts severe constraints on the physical properties of the gas.     

Phototropism, Adaptation, and the Light-Growth Response of Phycomyces

Phototropic bending can be initiated without the transient changes in growth speed that characterize a light-growth response. The conditions required are a change from a symmetric to an asymmetric illumination pattern while the cell receives a constant radiant flux. Phototropism is thus basically a steady state process. It cannot be founded on differential light-growth responses as in Blaauw's theory. A possible model system for the unequal partition of growth during steady bending is discussed. The fact that light-growth responses show adaptation while phototropic bending does not follows from the different natures of the two responses.     

Flavin-Binding Sites in Phycomyces

Abstract: Riboflavin-binding proteins could be the photorecep-tors for tropism in the fungus Phycomyces blakesleeanus . Radio-labelled riboflavin bound to both membrane-associated and cy-tosolic sites. The membrane sites (approximately 0.2–0.6nmol per g fresh material) were highly specific, with decreasing affinities for riboflavin (K_D≅ 1 μM under reducing conditions, K_D≅μM under oxidizing conditions), FMN, roseoflavin, and FAD. These binding sites, whose properties were similar to those of higher plants, could be solubilized with mild detergents, and were found in all vegetative parts of the fungus, including the spores. Mutants defective for phototropism did not differ from the wild type in the amount of binding sites or their affinity. A completely different binding to riboflavin was observed in the cytosolic supernatant of the sporangiophores; this activity was heat resistant and the binding sites could be partially purified and recognized as a polymerization product of gallic acid. Flavms were abundant in the sporangiophores (4.5 nmol per g fresh mass) and the spores (60 nmol per g fresh mass), but scarce in washed membranes (0.02–0.11 nmol per g fresh spor-angiophore mass). Autogenous fluorescence, whose absorption and emission wavelengths fit those of riboflavin, was seen by confocal microscopy, in part as clustered particles, in the actively growing parts of the mycelium, in the cytoplasm of sporangiophores, and in the spores.     

The Lens Effect and Phototropism of Phycomyces

Normally, the dioptrics in air of the cylindrical sporangiophore of Phycomyces blakesleeanus confer on the distal side a focusing advantage of about 30 per cent for unilateral stimuli of parallel light. This advantage can be nullified or reversed to produce negative curvatures by means of diverging light stimuli. A thin cylindrical glass lens was positioned 0.15 mm from the light-adapted growing zone with its long axis parallel to the long axis of the sporangiophore. A 3 minute blue stimulus was given and the lens removed. Reproducible negative curvatures were observed with a maximum of 13 degrees occurring within 8 minutes after the beginning of the stimulus. Experiments in air were done in a water-saturated atmosphere to minimize avoidance responses due to the proximity of the lens. The data support Buder's conclusion that the focusing advantage is the principal mechanism which produces the response differential necessary for phototropism. When the lens advantage is small, the attenuation becomes important in determining the direction of the response. Data obtained from sporangiophores immersed in inert liquids indicate that the attenuation is about 14 per cent. Therefore, whenever the focusing advantage is less than 14 per cent, negative curvatures are produced by unilateral stimuli.     

Phototropic Curvature in Phycomyces

The distribution of curvature and of bending speed along the cell's growing region are studied during steady state phototropic bending. At the start, elemental bending speed parallels the known axial distribution of growth rate. Hence regional phototropic sensitivity is initially determined by the local growth rate, and unilateral visible light acts proportionally at all levels of the growth zone. In the later course of bending, the bending speed distribution shifts downward instead of progressing upward in step with the cell's elongation. Furthermore, during phototropic inversion reversed bending begins high in the growth zone and progresses downward while normal bending continues below. These spatial and temporal changes in the distribution of differential growth are considered to be due to a fixed rate of supply of material used in growth that is transported from lower regions of the cell and asymmetrically distributed within the growth zone.     

Steady-State Phototropism in Phycomyces

The steady-state phototropic bending of Phycomyces sporangiophores was studied using apparatus designed to keep the growing zone vertical and the angle of illumination constant over long periods of time. The bending speed is quite constant if the intensity and angle of illumination are fixed. A phototropic inversion occurs in response to a sudden change in intensity, either an increase or a decrease. A bending component lateral to the illumination direction is strongly evident at normal incidence. It is shown that this component is due to a rotation between the stimulus and response loci about the axis of the growing zone, which is probably related to the spiral growth of the cell. The steady-state bending speed is at a maximum value for illumination directions ranging from normal incidence to about 45°. From 45 to 14° the bending speed decreases linearly with angle, reaching zero at 14°. Angles less than 14° elicit a weak negative phototropic response. Using an optical model of the growing zone, the intracellular intensity distribution was determined as a function of the angle of illumination. Several hypotheses relating the intensity distribution to the phototropic response are discussed.     

Bat Avoidance in Non-Aerial Insects: The Silence Response of Signaling Males in an Acoustic Moth

While the evasive responses of many flying acoustic insects to aerial‐hawking bats are duly recognized and studied, the responses of non‐aerial insects to gleaning bats are generally overlooked. It has been assumed that acoustic insects are deaf to these predators because gleaning bat echolocation calls are typically low in amplitude, brief (1–3 ms) and very high in frequency (>60 kHz). We tested this assumption in a series of playback experiments with a moth (Achroia grisella) that uses hearing in both predator evasion and mating. We report that ultrasound pulses ≥78 dB peSPL (peak equivalent sound pressure level) and ≥1 ms in duration inhibit stationary males from broadcasting their own ultrasonic advertisement calls, provided that the pulsed stimuli are delivered at a repetition rate ≤30/s. Further analyses suggest that inhibition by pulsed ultrasound comprises two processes performed serially. First, a startle response with a latency <50 ms is elicited by a single pulse ≥1 ms duration. Here, a male misses broadcasting several calls over a 50–100 ms interval. Secondly, the startle may be extended as a silence response lasting several to many seconds if subsequent pulses occur at a rate ≤30/s. Call inhibition cannot represent a simple response to acoustic power because of the inverse interaction between pulse duration and rate. On the other hand, the temporal and energy characteristics of inhibitory stimuli match those of gleaning bat echolocation calls, and we infer that inhibition is a specialized defensive behavior by which calling males may avoid detection by eavesdropping bats.     

Liposome-protoplast fusion in Phycomyces blakesleeanus

Abstract We describe here fusion between phospholipid vesicles (liposomes) and protoplasts to the fungus Phycomyces blakesleeanus . Both 6-carboxyfluorescein and the kanamycin resistance harboured by the plasmid have been transferred from liposomes to protoplasts of Phycomyces by the fusion technique.     

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, β-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in β-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of β-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress β-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.     

Carbamoyl-phosphate synthase in Phycomyces blakesleeanus

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.     

Uninucleate spores of Phycomyces

Summary Under most culture conditions only 0.3% of the vegetative spores of Phycomyces blakesleeanus are uninucleate. On an acidified minimal medium, the uninucleate fraction can be raised up to 4.5% of the spores. The spore population can be fractionated in a gradient under gravity (1xg) yielding fractions that contain over 80% uninucleate spores. These uninucleate spores are fully viable. When the spores to be fractionated are obtained from a heterokaryotic mycelium, the uninucleate fraction produces homokaryotic mycelia.