Helical growth during the phototropic response, avoidance response, and in stiff mutants of Phycomyces blakesleeanus.

The sporangiophores of Phycomyces blakesleeanus have been used as a model system to study sensory transduction, helical growth, and to establish global biophysical equations for expansive growth of walled cells. More recently, local statistical biophysical models of the cell wall are being constructed to better understand the molecular underpinnings of helical growth and its behavior during the many growth responses of the sporangiophores to sensory stimuli. Previous experimental and theoretical findings guide the development of these local models. Future development requires an investigation of explicit and implicit assumptions made in the prior research. Here, experiments are conducted to test three assumptions made in prior research, that (a) elongation rate, (b) rotation rate, and (c) helical growth steepness, R, of the sporangiophore remain constant during the phototropic response (bending toward unilateral light) and the avoidance response (bending away from solid barriers). The experimental results reveal that all three assumptions are incorrect for the phototropic response and probably incorrect for the avoidance response but the results are less conclusive. Generally, the experimental results indicate that the elongation and rotation rates increase during these responses, as does R, indicating that the helical growth steepness become flatter. The implications of these findings on prior research, the "fibril reorientation and slippage" hypothesis, global biophysical equations, and local statistical biophysical models are discussed.

The inhibition of mating in Phycomyces blakesleeanus by light is dependent on the MadA-MadB complex that acts in a sex-specific manner.

Light is an environmental signal that influences reproduction in the Mucoromycotina fungi, as it does in many other species of fungi. Mating in Phycomyces blakesleeanus is inhibited by light, but the molecular mechanisms for this inhibition are uncharacterized. In this analysis, the role of the light-sensing MadA-MadB complex in mating was tested. The MadA-MadB complex is homologous to the Neurospora crassa White Collar complex. Three genes required for cell type determination in the sex locus or pheromone biosynthesis are transcriptionally-regulated by light and are controlled by MadA and MadB. This regulation acts through the plus partner, indicating that the inhibitory effect of light on mating is executed through only one of the two sexes. These results are an example whereby the mating types of fungi have acquired sex-specific properties beyond their role in conferring cell-type identity, and provide insight into how sex-determining chromosomal regions can expand the traits they control.

Growth inhibition of fungus Phycomyces blakesleeanus by anion channel inhibitors anthracene-9-carboxylic and niflumic acid attained through decrease in cellular respiration and energy metabolites.

Increasing resistance of fungal strains to known fungicides has prompted identification of new candidates for fungicides among substances previously used for other purposes. We have tested the effects of known anion channel inhibitors anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) on growth, energy metabolism and anionic current of mycelium of fungus Phycomyces blakesleeanus. Both inhibitors significantly decreased growth and respiration of mycelium, but complete inhibition was only achieved by 100 and 500 µM NFA for growth and respiration, respectively. A9C had no effect on respiration of human NCI-H460 cell line and very little effect on cucumber root sprout clippings, which nominates this inhibitor for further investigation as a potential new fungicide. Effects of A9C and NFA on respiration of isolated mitochondria of P. blakesleeanus were significantly smaller, which indicates that their inhibitory effect on respiration of mycelium is indirect. NMR spectroscopy showed that both A9C and NFA decrease the levels of ATP and polyphosphates in the mycelium of P. blakesleeanus, but only A9C caused intracellular acidification. Outwardly rectifying, fast inactivating instantaneous anionic current (ORIC) was also reduced to 33±5 and 21±3 % of its pre-treatment size by A9C and NFA, respectively, but only in the absence of ATP. It can be assumed from our results that the regulation of ORIC is tightly linked to cellular energy metabolism in P. blakesleeanus, and the decrease in ATP and polyphosphate levels could be a direct cause of growth inhibition.

(51)V NMR investigation of cell-associated vanadate species in Phycomyces blakesleeanus mycelium.

(51)V NMR spectroscopy was used for detection and identification of cell-associated vanadate (V(5+)) species after exposure of Phycomyces blakesleeanus mycelium, in exponential phase of growth, to sodium orthovanadate. Complete disappearance of monomer and dimer signals and decreased intensity of the tetramer signal were observed about 40 min after treatment. Simultaneously, a signal at -532 ppm, with increasing intensity, was detected in spectra. The time-dependent rise in this signal was connected to a decrease in the extracellular monomer signal, indicating its transport into the cell. The signal at -532 ppm did not belong to any known simple oxido-vanadate species, nor to a complex with any of the components of experimental medium. This signal was the only one present in spectrum of the mycelium washed 35 min after treatment, and the only one observed in mycelium cultivated on vanadate-contained medium. Therefore, its appearance can be attributed to intracellular complexation, and may represent an important detoxification mechanism of the cell exposed to a physiologically relevant concentration of vanadate. Experiments ((51)V NMR and polarography) performed with Cd-pretreated mycelium (inhibitor of an enzyme responsible for V(5+) reduction) and ferricyanide-preincubated mycelium excluded the possibility of V(5+) tetramer's entry into the cell.

A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae.

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

Stress in Phycomyces blakesleeanus by glucose starvation and acetate growth: response of the antioxidant system and reserve carbohydrates.

The objective of this study was to analyze the response of Phycomyces blakesleeanus to glucose starvation and acetate growth stress. At the onset of the exponential growth phase, the fungus shows a high tolerance to both stresses, being higher for the glucose starvation. In both stresses we have found higher activities of catalase and glutathione peroxidase, and a decrease of the pools of D-erythroascorbate (D-erythroascorbate+D-erythroascorbate monoglucoside) and glutathione (GSH+GSSG), while the intracellular GSH/GSSG redox balance becomes more reducing. Gallic acid was not detected under both stresses. Glycogen breakdown and the high levels of trehalose seem to be part of the stress response. Both stress, under the conditions of this study, seem to lead to a qualitatively similar response in P. blakesleeanus, with regard to the behavior of antioxidant system, the content of secondary metabolites and the role of the reserve carbohydrates.

The sporangiophore of Phycomyces blakesleeanus: a tool to investigate fungal gravireception and graviresponses.

The giant sporangiophore of the single-celled fungus, Phycomyces blakesleeanus, utilises light, gravity and gases (water and ethylene) as environmental cues for spatial orientation. Even though gravitropism is ubiquitous in fungi (Naturwissenschaftliche Rundschau, 1996, 49, 174), the underlying mechanisms of gravireception are far less understood than those operating in plants. The amenability of Phycomyces to classical genetics and the availability of its genome sequence makes it essential to fill this knowledge gap and serve as a paradigm for fungal gravireception. The physiological phenomena describing the gravitropism of plants, foremost adherence to the so-called sine law, hold even for Phycomyces. Additional phenomena pertaining to gravireception, specifically adherence to the novel exponential law and non-adherence to the classical resultant law of gravitropism, were for the first time investigated for Phycomyces. Sporangiophores possess a novel type of gravisusceptor, i.e. lipid globules that act by buoyancy rather than sedimentation and that are associated with a network of actin cables (Plant Biology, 2013). Gravitropic bending is associated with ion currents generated by directed Ca(2+) and H(+) transport in the growing zone (Annals of the New York Academy of Sciences, 2005, 1048, 487; Planta, 2012, 236, 1817). A set of behavioural mutants with specific defects in gravi- and/or photoreception allowed dissection of the respective transduction chains. The complex phenotypes of these mutants led to abandoning the concept of simple linear transduction chains in favour of interacting networks with molecular modules of physically interacting proteins.

Actin cytoskeleton and organelle movement in the sporangiophore of the zygomycete Phycomyces blakesleeanus.

Growth, photo- and gravitropism of sporangiophores of the zygomycete Phycomyces blakesleeanus occur within the apical growing zone, a cylindrical structure (diameter about 100 µm) that reaches about 1.5-2.5 mm below the tip and has growth rates up to 50 µm·min(-1) . To better understand morphogenesis and growth of the giant aerial hypha, we investigated with confocal microscopy and inhibitors the actin cytoskeleton and by in-vivo particle tracking the associated organelle movement. We found stage-1 sporangiophores (without sporangium) possess an actin cytoskeleton with polar zonation. (i) In the apex, abundant microfilaments without preferential orientation entangled numerous nuclei as well as a conspicious complex of some 200 lipid globules. Microfilament patches (≈ 1.6-µm diameter) are clustered in the tip and were found in the apical cortex, whereas short, curved microfilament bundles (≈ 2.3-µm long) prevailed in the subapex. (ii) In a transition zone downwards to the shaft, the microfilaments rearranged into a dense mat of longitudinal microfilaments that was parallel close to the periphery but more random towards the cell centre. Numerous microfilament patches were found near the cortex (≈ 10/100 µm(2) ); their number decreased rapidly in the subcortex. In contrast, the short, curved microfilament bundles were found only in the subcortex. (iii) The basal shaft segment of the sporangiophore (with central vacuole) exhibited bidirectional particle movement over long distances (velocity ≈ 2 µm·s(-1) ) along massive longitudinal, subcortical microfilament cables. The zonation of the cytoskeleton density correlated well with the local growth rates at the tip of the sporangiophore, and appears thus as a structural prerequisite for growth and bending.

Antioxidant defence system during exponential and stationary growth phases of Phycomyces blakesleeanus: response to oxidative stress by hydrogen peroxide.

An analysis of the components of the antioxidant defence system in exponential and stationary growth phases of filamentous fungus Phycomyces blakesleeanus and the response to the oxidative stress hydrogen peroxide were performed. There is a strong positive correlation between mycelial antioxidant capacity and the contents of gallic acid, d-erythroascorbate (d-EAA) or d-erythroascorbate monoglucoside (d-EAAG). These secondary metabolites are specifically synthesized by this fungus and reach maximal values in the stationary growth phase, suggesting that they can play some role in the antioxidant defence system of this fungus. There is a differential expression of the two more notable antioxidant activities, catalase (CAT) and superoxide dismutase (SOD), depending of the growth stage of P. blakesleeanus, CAT being expressed in the exponential and SOD in the stationary phase. Phycomyces blakesleeanus showed a high resistance to the oxidative stress caused by H2O2 (50 and 200 mM) which was higher in exponential phase. This higher resistance can be explained by the presence of CAT, glutathione peroxidase (GPx), and the probable contribution of glutathione-S-transferase (GST) and high levels of reduced form of glutathione (GSH). The transition to stationary phase was accompanied with a higher physiological oxidative damage illustrated by the higher protein carbonylation. In this growth stage the resistance of the fungus to the oxidative stress caused by H2O2 could be explained by the presence of SOD, GPx, and the probable contribution of GST as well as of secondary metabolites, mainly d-EAA and d-EAAG. These results highlight a specific response to oxidative stress by H2O2 depending on the growth phase of P. blakesleeanus.

Surface tip-to-base Ca2+ and H+ ionic fluxes are involved in apical growth and graviperception of the Phycomyces stage I sporangiophore.

Net fluxes of Ca(2+) and H(+) ions were measured non-invasively close to the surface of Phycomyces blakesleeanus sporangiophores stage I using ion-selective vibrating microelectrodes. The measurements were performed on a wild type (Wt) and a gravitropic mutant A909 kept in either vertical or tilted orientation. Microelectrodes were positioned 4 µm from the surface of sporangiophore, and ion fluxes were recorded from the apical (0-20 µm) and subapical (50-100 µm) regions. The magnitude and direction of ionic fluxes measured were dependent on the distance from the tip along the growing zone of sporangiophore. Vertically oriented sporangiophores displayed characteristic tip-to-base ion fluxes patterns. Ca(2+) and H(+) fluxes recorded from apical region of Wt sporangiophores were inward-directed, while ion fluxes from subapical locations occurred in both directions. In contrast to Wt, mutant A909 showed opposite (outward) direction of Ca(2+) fluxes and reduced H(+) influxes in the apical region. Following gravistimulation, the magnitude and direction of ionic fluxes were altered. Wt sporangiophore exhibited oppositely directed fluxes on the lower (influx) and the upper (efflux) sides of the cell, while mutant A909 did not show such patterns. A variable elongation growth in vertical position and reduced growth rate upon gravistimulation were observed in both strains. The data show that tip-growing sporangiophores exhibit a tip-to-base ion flux pattern which changes characteristically upon gravistimulation in Wt in contrast to the mutant A909 with a strongly reduced gravitropic response.

Spontaneous rotational inversion in Phycomyces.

The filamentary fungus Phycomyces blakesleeanus undergoes a series of remarkable transitions during aerial growth. During what is known as the stage IV growth phase, the fungus extends while rotating in a counterclockwise manner when viewed from above (stage IVa) and then, while continuing to grow, spontaneously reverses to a clockwise rotation (stage IVb). This phase lasts for 24-48 h and is sometimes followed by yet another reversal (stage IVc) before the overall growth ends. Here, we propose a continuum mechanical model of this entire process using nonlinear, anisotropic, elasticity and show how helical anisotropy associated with the cell wall structure can induce spontaneous rotation and, under appropriate circumstances, the observed reversal of rotational handedness.

Glycogen and trehalose mobilization by acetic acid in Phycomyces blakesleeanus: dependence on the anion form.

Trehalose and glycogen are reserve carbohydrates that were shown to accumulate in mycelia of the filamentous fungus Phycomyces blakesleeanus. Both carbohydrates were mobilized under glucose starvation or in the presence of acetate. Glycogen was mobilized faster than trehalose in the presence of acetate. In all cases, glycogen and trehalose mobilization followed single exponential decay. There was a direct relationship between glycogen mobilization and the concentration of the dissociated form of external acetic acid. The half-life of glycogen mobilization increased as the concentration of the external acetate anion decreased, so the dissociated form of acetate was the stressor causing glycogen mobilization. Mobilization was not due to transfer to poor carbon sources, as the dissociated form of other weak acids (butyrate, lactate, pyruvate and propionate) also produced glycogen mobilization. Previous exposure of the mycelia to a lower acetate concentration decreased glycogen mobilization by subsequent exposure to a high acetate concentration. Glycogen mobilization by acetate may be involved in production of ATP necessary for acetate uptake as well as for maintenance of the internal pH homeostasis.

Ubiquinone and carotene production in the Mucorales Blakeslea and Phycomyces.

The filamentous fungi Phycomyces blakesleeanus and Blakeslea trispora (Zygomycota, Mucorales) are actual or potential industrial sources of beta-carotene and lycopene. These chemicals and the large terpenoid moiety of ubiquinone derive from geranylgeranyl pyrophosphate. We measured the ubiquinone and carotene contents of wild-type and genetically modified strains under various conditions. Light slightly increased the ubiquinone content of Blakeslea and had no effect on that of Phycomyces. Oxidative stress modified ubiquinone production in Phycomyces and carotene production in both fungi. Sexual interaction and mutations in both organisms made the carotene content vary from traces to 23 mg/g dry mass, while the ubiquinone content remained unchanged at 0.3 mg/g dry mass. We concluded that the biosyntheses of ubiquinone and carotene are not coregulated. The specific regulation for carotene biosynthesis does not affect even indirectly the production of ubiquinone, as would be expected if terpenoids were synthesized through a branched pathway that could divert precursor flows from one branch to another.

31P NMR study of polyphosphate levels during different growth phases of Phycomyces blakesleeanus.

The changes in relative polyphosphate content, estimated as the intensity ratio of core polyphosphate signal and intracellular inorganic phosphate signal from 31P NMR spectra, during the growth of Phycomyces blakesleeanus are reported. The ratio increases from 16 h to 28 h of growth, the minimum occurs at 32 h, followed by sharp increase up to 36 h, and a steady decrease afterwards. The changes in the biomass during mycelium growth showed steady increases, with a stagnation period between 32 h and 36 h during which a pronounced increase in the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal occurred. The reduction of growth temperature from 22 degrees C to 18 degrees C significantly decreased the rate and intensity of growth, but the pattern of polyphosphate changes remained unchanged. The changes of the intensity ratio of core polyphosphates to intracellular inorganic phosphate signal are linked to characteristic stages of sporangiophore development. Analysis of core polyphosphates, intracellular inorganic phosphate and beta-ATP signal intensities suggest the role of polyphosphates as an energy and/or a phosphate reserves during Phycomyces development.

Modification of sexual development and carotene production by acetate and other small carboxylic acids in Blakeslea trispora and Phycomyces blakesleeanus.

In Phycomyces blakesleeanus and Blakeslea trispora (order Mucorales, class Zygomycetes), sexual interaction on solid substrates leads to zygospore development and to increased carotene production (sexual carotenogenesis). Addition of small quantities of acetate, propionate, lactate, or leucine to mated cultures on minimal medium stimulated zygospore production and inhibited sexual carotenogenesis in both Phycomyces and Blakeslea. In Blakeslea, the threshold acetate concentration was <1 mmol/liter for both effects, and the concentrations that had one-half of the maximal effect were <2 mmol/liter for carotenogenesis and >7 mmol/liter for zygosporogenesis. The effects on Phycomyces were similar, but the concentrations of acetate had to be multiplied by ca. 3 to obtain the same results. Inhibition of sexual carotenogenesis by acetate occurred normally in Phycomyces mutants that cannot use acetate as a carbon source and in mutants whose dormant spores cannot be activated by acetate. Small carboxylic acids may be signals that, independent of their ability to trigger spore germination in Phycomyces, modify metabolism and development during the sexual cycle of Phycomyces and Blakeslea, uncoupling two processes that were thought to be linked and mediated by a common mechanism.

Ca2+ and H+ ion fluxes near the surface of gravitropically stimulated Phycomyces sporangiophore.

The single-celled fungus Phycomyces blakesleeanus forms vertically oriented sporangiophores from hyphae, which display a negative gravitropic response. Longitudinal growth and gravitropic bending of these sporangiophores were measured with noninvasive H(+) and Ca(2+) ion-selective microelectrode measurements. The directions of H(+) and Ca(2+) fluxes, recorded at different locations of sporangiophores, were opposite when the sporangiophores were kept in vertical position. Ca(2+) fluxes were in most experiments positive (efflux), while H(+) fluxes were negative (influx). The direction of ion fluxes depended on developmental stage of sporangiophores and changed with gravistimulation.

Genetic damage following introduction of DNA in Phycomyces.

Introduction of plasmids in Phycomyces blakesleeanus caused extensive changes in the exogenous DNA and in the resident genome. Plasmids with a bacterial gene for geneticin resistance under a Phycomyces promoter were either injected into immature sporangia or incubated with spheroplasts. An improved method produced about one viable spheroplast per cell. Colonies resistant to geneticin were rare and only about 0.1% of their spores grew in the presence of geneticin. The transformation frequency was very low, < or =1 transformed colony per million spheroplasts or per microg DNA. Few nuclei in the transformants contained exogenous DNA, as shown by a selective procedure that sampled single nuclei from heterokaryons. The exogenous DNA was not integrated into the genome and no stable transformants were obtained. The plasmids were replicated in the recipient cells, but their DNA sequences were modified by deletions and rearrangements and the transformed phenotype was eventually lost. The spores developed in injected sporangia were often inviable; a genetic test showed that spore death was caused by impaired nuclear proliferation and induction of lethal mutations. About one-fourth of the viable spores from injected sporangia formed abnormal colonies with obvious changes in shape, texture, or color. The abnormalities that could be investigated were due to dominant mutations. The results indicate that incoming DNA is not only attacked, but signals a situation of stress that leads to increased mutation and nuclear and cellular death.

Quickly changing acceleration forces (QCAFs) vibration analysis on the A300 ZERO-G.

Experiments that are done under microgravity, e.g. during space or parabola flights, are invariably accompanied and affected by ubiquitous vibrations of the surroundings. Vibrations induce Quickly Changing Acceleration Forces (QCAFs) that interfere with the perception of the earth gravitational field. To investigate their impact on experiments under microgravity we monitored the vibrations of the airplane A300 ZERO-G during parabola flights and analyzed them in their spectral and frequency domains. Power spectra obtained with Fast-Fourier Transforms (FFT) display a complex pattern of various vibrations whose origin, relative phases and intensities remain unidentified. During the zero-g phases (parabolas), when the engines of the airplane are throttled, the vibrations still elicit residual QCAFs of at least +/- 1 g. By means of adequate damping procedures the QCAFs could, however, be reduced by approximately 95%.

Analysis of phenomena involved in the apical growth of Phycomyces blakesleeanus.

The effects of the Ca(2+)/H(+) exchanger A23187 and the K(+)/H(+) exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca(2+) channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.

Helical growth of stage-IVb sporangiophores of Phycomyces blakesleeanus: the relationship between rotation and elongation growth rates.

An understanding of the relationship between the two components of helical growth (rotation rate and elongation rate) is fundamental to understanding the biophysical and molecular mechanism(s) of cell wall extension in algal cells, fungal cells, and plant stems and roots. Helical growth occurs throughout development of the sporangiophores of Phycomyces blakesleeanus. Previous studies within the growth zone of stage-IVb sporangiophores have reported conflicting conclusions. An implicit assumption in the previous studies [E.S. Castle (1937) J Cell Comp Physiol 9:477-489; R. Cohen and M. Delbruck (1958) J Cell Comp Physiol 52:361-388; J.K.E. Ortega et al. (1974) Plant Physiol 53:485-490] was that the relationship between rotation rate and elongation rate was independent of the magnitude of the elongation rate. In the present study, for stage-IVb sporangiophores growing at a steady rate, it is shown that the ratio of rotation rate and elongation rate decreases as the elongation rate increases. Previously proposed biophysical and molecular mechanisms cannot account for the observed behavior. The previously postulated fibril-reorientation mechanism [J.K.E. Ortega and R.I. Gamow (1974) J Theor Biol 47:317-332; J.K.E. Ortega et al. (1974) Plant Physiol 53:485-490] is modified to accommodate this new finding. Other experiments were conducted to determine how the ratio of rotation rate and elongation rate behaves during a pressure response (a transient decrease in elongation rate produced by a large step-up in turgor pressure using the pressure probe). Results of these experiments indicate that this ratio increases during the pressure response.