Phycomyces: a change in mechanical properties after a light stimulus

Tensile tests were conducted on the photoresponsive stage IVb sporangiophore of the fungus Phycomyces before and after a saturating light stimulus. The results demonstrate that an increase in the mechanical extensibility of the cell wall occurs after the light stimulus. This increase in mechanical extensibility occurs in the growing zone of the sporangiophore. The majority of this increase occurs in the region about 300 ųmeters beneath the sporangium.     

“Apical dominance” in the sporangiophore of the fungus Phycomyces

Summary Control, by the spores, over the elongation and the branching of the sporangiophore is described. If the sporangium is removed from a sporangiophore, the sporangiophore stops growing within a few hours. 6–16h later a branch grows from the defunct growing zone. This branch forms a new sporangium and spores, and then starts to elongate again. The original growing zone can be rescued by replacing the sporangium with another sporangium or a sporangium-sized drop of spores. If the original growing zone is rescued, then it continues growing and the sporangiophore does not form a branch. It can only be rescued, however, within the first 60 min after the original sporangium is removed.This work was submitted in partial fulfillment of the requirements for the Ph. D. degree.     

Analysis of growth and rotational behavior of sporangiophores in Pilobolus crystallinus (Wiggers) Tode

The growth and rotation of the sporangiophore of Pilobolus crystallinus, which are important factors in its phototropic behavior, were analyzed throughout its development. The sporangiophore initial emerged from the trophocyst and elongated at the extreme tip without rotating. The elongation rate of the sporangiophore apex then gradually decreased and the apex expanded radially to produce the sporangium, but no rotation occurred. A transient cessation of elongation after sporangium development was followed by resumption of both elongation and radial expansion in the region beneath the sporangium developing the subsporangial vesicle. Rotation was not obvious at this stage. Radial expansion of the subsporangial vesicle continued at a decreasing rate until full size was reached. Elongation then recommenced in the newly established growth zone in the upper region of the sporangiophore just beneath the subsporangial vesicle. During this period of growth, the sporangiophore rotated in a clockwise direction as viewed from above. All growth and rotation ceased about 1 h before ejection of the sporangium into the air. Based on these results, a modified classification of the developmental stages has been proposed.This work was carried out under the Joint Research Program of the Institute of Genetic Ecology, Tohoku University, Japan (892006). The authors please to thank Kaori Koga and Hiroko Kikuchi for their helpful assistance.     

Phycomyces: MODIFICATION OF LIGHT-INDUCED SPIRAL GROWTH AFTER MECHANICAL CONDITIONING OF THE CELL WALL

Mature stage IVb Phycomyces sporangiophores show left-hand spiral growth; that is, viewed from above, the sporangium rotates clockwise. It has been shown that mechanical conditioning (strain-hardening) of the cell wall by the Instron technique increases the ratio of rotation to the elongation growth rate compared to nonmechanically conditioned controls. It is reported that the addition of a saturating light stimulus to these sporangiophores causes a decrease in the ratio of rotation to elongation growth rate. This result is in agreement with the fibril slippage model, i.e. the counterclockwise rotation of stage IVa is a result of parallel fibrils lying in a right-handed spiral configuration slipping by one another. It is suggested that a light stimulus added to a mechanically conditioned stage IVb sporangiophore activates one or more cell wall-loosening enzymes which act by decreasing the number of intermolecular bonds between parallel fibrils causing fibril slippage, resulting in counterclockwise rotation. It is precisely this counterclockwise contribution that decreases the rotation to elongation growth ratio of mechanically conditioned and then light-stimulated stage IVb sporangiophores.     

Photo-reactions in Phycomyces; growth and tropic responses to the stimulation of narrow test areas

Sporangiophores of Phycomyces in stage IV b have been stimulated by parallel light in test areas 0.2 mm. wide. The growth responses to large stimuli are very large, owing probably to light scattered within the specimen. For medium stimuli the sensitive zone coincides with the growth response zone obtained previously and excludes the region of maximum stretch. Sustained stimulations were used to elicit tropic responses. The bends formed travel away from the sporangium at a speed equal to the growth speed. Thus they remain very close to the stimulus when this is held at a constant level relative to ground but separate from it for stimuli programmed differently. The existence of a protoplasmic structure, the "inner wall," with the following properties is postulated: it is attached to the lower, non-growing part of the sporangiophore and grows by addition above the sensitive zone. It neither stretches nor twists in the sensitive zone. It is the seat of the light receptors and gives growth and tropic responses. The cell wall follows its bends by elastic stretch.     

Phycomyces: fine structure analysis of the growing zone

Fine structure analysis of the stage IVb Phycomyces sporangiophore growing zone (GZ) was performed during steady-state growth using a computer-video digitizer and recorder. By simultaneously measuring the trajectory of two independent particles above and within the GZ, we have confirmed the previous findings of R. Cohen and M. Delbrück (1958 J Cell Comp Physiol 52: 361-388) that the GZ is not uniform. We have been unable to confirm their findings that counterclockwise rotation exists in a mature sporangiophore. The rates of rotation and elongation change independently as a function of position in the GZ. This change is not linear as would be expected if the GZ were uniform. The importance of this finding is discussed in terms of the fibril reorientation model.     

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.     

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.     

Intracellular recordings from phycomyces

Intracellular recordings from the giant sporangiophore of Phycomyces stage II were obtained. The mean transmembrane potential for 30 observations was −119.9 millivolts (negative inside), and it did not change either as a result of a light stimulus or during dark adaptation. Injected depolarizing and hyperpolarizing step currents and steady currents did not produce any avidence of spike activity. We conclude that light transduction and dark adaptation in Phycomyces are not based on alterations of the transmembrane potential.     

Cell wall extension behavior of Phycomyces sporangiophores during the pressure response.

The cylindrical, single-celled sporangiophore of Phycomyces blakesleeanus grows (enlarges) predominantly in the longitudinal direction during two stages of development; stage I and stage IVb. Cell enlargement (cell wall extension) occurs in a distinct region termed the "growing zone." It was previously reported that a large step-up or pulse-up in turgor pressure, greater than approximately 0.02 MPa, will elicit a transient decrease in longitudinal growth rate of the stage I and stage IVb sporangiophore. This transient decrease in longitudinal growth rate is termed the "pressure response." Both the magnitude and duration of the pressure response depend on the magnitude of the turgor pressure step-up or pulse-up. Qualitatively, the pressure response is similar to the stretch response, which is produced with the application of a longitudinal force (load) on the sporangiophore. In this investigation, the growth (extension) behavior of the cell wall in the growing zone is studied during the pressure response. It is found that both the extension rate of the cell wall in the growing zone and the length of the growing zone decrease during the pressure response, and that together they account for the observed decrease in longitudinal growth rate.     

Growth rate fluctuations in phycomyces sporangiophores

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input (“night-blind”) or output (“stiff” and “hypertropic”) of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.     

Tropic Responses of Phycomyces Sporangiophores to Gravitational and Centrifugal Stimuli

A low-speed centrifuge was used to study the tropic responses of Phycomyces sporangiophores in darkness to the stimulus of combined gravitational and centrifugal forces. If this stimulus is constant the response is a relatively slow tropic reaction, which persists for up to 12 hours. The response is accelerated by increasing the magnitude of the gravitational-centrifugal force. A wholly different tropic response, the transient response, is elicited by an abrupt change in the gravitational-centrifugal stimulus. The transient response has a duration of only about 6 min. but is characterized by a high bending speed (about 5°/min.). An analysis of the distribution of the transient response along the growing zone shows that the active phase of the response has a distribution similar to that of the light sensitivity for the light-growth and phototropic responses. Experiments in which sporangiophores are centrifuged in an inert dense fluid indicate that the sensory mechanism of the transient response is closely related to the physical deformation of the growing zone caused by the action of the gravitational-centrifugal force on the sporangiophore as a whole. However, the response to a steady gravitational-centrifugal force is most likely not connected with this deformation, but is probably triggered by the shifting of regions or particles of differing density relative to one another inside the cell.     

The Plasmodium Class XIV Myosin,MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic “head,” a modified “neck,” and the absence of a “tail” region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.     

A quantitative model of Phycomyces phototropism

A quantitative model accounting for phototropism in the wild type and in behavioural mutants of Phycomyces is described.Photomecisms (changes in the sporangiophore's growth velocity in response to changes in light intensity) are produced by a system composed of two sets of linear transducers separated by an adaptation mechanism, the first transducer being the photoreceptor.Phototropism under asymmetrical light distributions is caused by the summation of local photomecisms in the distal half of the sporangiophore, where two bright bands are produced by refraction of the incident light. The photoreceptors turn around the sporangiophore axis; they are approximately adapted to local intensity everywhere except upon entrance to the first bright band. Thus, a continuous photomecism originates at this band while the rest of the sporangiophore remains practically unstimulated.The mutants suffer a reduction in the efficiency of transduction.The behaviour of the wild type and of the mutants has been quantitatively simulated by computer. The predictions from the model fit the experimental results.     

Photoreactions in Phycomyces : Responses to the stimulation of narrow test areas with ultraviolet light

Equipment has been developed for ultraviolet illumination of sharply bounded test areas of the growing zone of sporangiophores of Phycomyces. The growing zone is opaque for this light and the tropic responses are negative. Periodic short narrow stimuli on alternating sides produce periodic tropic responses when applied at x > 0.5 mm, but none for x < 0.5 mm, where x is the distance below the sporangium. Sustained tropic stimuli, applied at constant x, produce tropic responses for any x > 0.1 mm. The lag between stimulus and response is 3.3 min. for any x > 0.5 mm. For smaller x the lag increases progressively. In all cases the tropic bend occurs at values of x > 0.5 mm. Sustained tropic stimuli, applied at constant height relative to ground, produce relatively sharp tropic bends. The center of the bend is at all times close to the simultaneous position of the stimulated area. The boundaries of a light-adapted zone move less than 0.1 mm in 10 min. relative to the sporangium. It is concluded that the receiving and adapting structures do not move relative to the sporangium, and that the responding system does not move relative to ground. The two systems move relative to each other with the speed of growth. The responding system does not extend above x = 0.5 mm.     

A Semidian Rhythm in the Flowering Response of Pharbitis nil to Far-Red Light: I. Phasing in Relation to the Light-Off Signal

Evidence is presented of an endogenous rhythm in flowering response to far-red (FR) irradiation, with a period of about 12 h (hence semidian rhythm), which persists through at least three cycles in constant conditions of continuous light at 27°C and has a marked influence on the flowering response in Pharbitis nil to a subsequent inductive dark period. The phase of the rhythm is not influenced by real time nor by the time from imbibition or from the beginning of the light period. Rather, it is fed forward from the beginning of the FR interruption to the beginning of the inductive dark period. The period of the rhythm is not affected by irradiance but is longer at cooler temperature. When there are two FR interruptions during the preceding light period, it is primarily the later one which determines the phase of the rhythm, although some interactions are evident. There appears to be an abrupt rephasing of the rhythm at the beginning of the inductive dark period. No overt rhythms which could be used as “clock hands” for the semidian rhythm were detected in photosynthesis, stomatal opening, or translocation.     

The Distribution of Cytoplasmic Vesicles, Multivesicular Bodies and Paramural Bodies in Elongating Sporangiophores and Swelling Sporangia of Thamnidium elegans Link

In Thamnidium elegans Link, cytoplasmic vesicles of variablesize were present in large numbers in sporangiophore apicesin agar and in smaller numbers in sporangiophore apices in air.Golgi-like cisternae were associated with small, sub-apicalclusters of vesicles. Apices in agar contained a nucleus andmitochondrion-free tip zone in which vesicles were concentrated.This zone was almost completely absent from apices in air. Inswelling sporangia, vesicles were sparce and were not concentratedagainst the wall. Rates of surface area increase were similarfor sporangiophores elongating, respectively, in agar and inair. Rates of surface area increase during sporangium swellingwere equal to or greater than rates of surface area increaseduring sporangiophore elongation. Vesicles were associated withformation of a secondary wall layer in swollen sporangia. Paramuralbodies and multivesicular bodies were present at all stagesof sporangiophore elongation and sporangium swelling. Isolatedhalo bodies (apical corpuscles) were present in walls at sporangiophoretips, and clusters of similar bodies were present in side wallsof sporangiophores.     

Mortierella oedorhiza,a new species forming a dichotomously branched rhizoid at the sporangiophore base

A new species, Mortierella oedorhiza, is described as a fungus forming a dichotomously branched rhizoid with a bulbil-like mass of hyphae at the base of the sporangiophore. The species is morphologically and phylogenetically similar to M. rostafinskii, which has rarely been reported. A trace of a columella on dehiscence of the sporangium and a dichotomously branched rhizoid are distinguishing characteristics of this species compared with M. rostafinskii.     

DARK ADAPTATION AND THE LIGHT-GROWTH RESPONSE OF PHYCOMYCES

1. A single-celled, elongating sporangiophore of Phycomyces responds to a sufficient increase in intensity of illumination by a brief increase in growth rate. This is the "light-growth response" of Blaauw. 2. The reaction time is compound, consisting of an exposure period and a latent period (this comprising both the true latent period resulting from photochemical action and any "action time" necessary for the response). During the latter period the plant may be in darkness, responding nevertheless at the end of the latent period. 3. Both light adaptation and dark adaptation occur in the sporangiophore. The kinetics of dark adaptation can be accounted for on the basis of a bimolecular reaction, perhaps modified by autocatalysis. Attention is called to the bimolecular nature of the "dark" reaction in all other photosensory systems that have been studied, in spite of the diversity of the photosensitive substances themselves and of the different forms of the responses to light.     

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.