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.

Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides.

The transcription of about 5-10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA-D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi.

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.

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.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

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.

The Uve1 endonuclease is regulated by the white collar complex to protect cryptococcus neoformans from UV damage.

The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome.

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.

Gravity-induced absorption changes in Phycomyces blakesleeanus during parabolic flights: first spectral approach in the visible.

Gravity-induced absorption changes as experienced during a series of parabolas on the Airbus 300 Zero-G have been measured previously pointwise on the basis of dual-wavelength spectroscopy. Only the two wavelengths of 460 and 665 nm as generated by light-emitting diodes have been utilised during our first two parabolic-flight campaigns. In order to gain complete spectral information throughout the wavelength range from 400 to 900 nm, a miniaturized rapid scan spectrophotometer was designed. The difference of spectra taken at 0 g and 1.8 g presents the first gravity-induced absorption change spectrum measured on wild-type Phycomyces blakesleeanus sporangiophores, exhibiting a broad positive hump in the visible range and negative values in the near infrared with an isosbestic point near 735 nm. The control experiment performed with the stiff mutant A909 of Phycomyces blakesleeanus does not show this structure. These results are in agreement with those obtained with an array spectrophotometer. In analogy to the more thoroughly understood so-called light-induced absorption changes, we assume that gravity-induced absorption changes reflect redox changes of electron transport components such as flavins and cytochromes localised within the plasma membrane.

The Phycomyces madA gene encodes a blue-light photoreceptor for phototropism and other light responses.

Phycomyces blakesleeanus is a filamentous zygomycete fungus that produces striking elongated single cells that extend up to 10 cm into the air, with each such sporangiophore supporting a sphere containing the spores for dispersal. This organism has served as a model for the detection of environmental signals as diverse as light, chemicals, touch, wind, gravity, and adjacent objects. In particular, sporangiophore growth is regulated by light, and it exhibits phototropism by bending toward near-UV and blue wavelengths and away from far-UV wavelengths in a manner that is physiologically similar to plant phototropic responses. The Phycomyces madA mutants were first isolated more than 40 years ago, and they exhibit reduced sensitivity to light. Here, we identify two (duplicated) homologs in the White Collar 1 family of blue-light photoreceptors in Phycomyces. We describe that the madA mutant strains contain point mutations in one of these genes and that these mutations cosegregate with a defect in phototropism after genetic crosses. Thus, the phototropic responses of fungi through madA and plants through phototropin rely on diverse proteins; however, these proteins share a conserved flavin-binding domain for photon detection.

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.

The gene for the heat-shock protein HSP100 is induced by blue light and heat-shock in the fungus Phycomyces blakesleeanus.

We cloned and sequenced the Phycomyces hspA gene. The hspA gene product is a 901-amino-acid protein member of the clpB/HSP100 family. HSP100 proteins are ATPases involved in high-temperature tolerance, proteolysis, and protein disaggregation. Phycomyces HSP100 is composed of a domain presumably involved in protein-protein interactions and two ATP-binding domains. The hspA promoter contains three heat-shock elements that are presumably involved in the activation of hspA after heat-shock. In addition, four short sequences are present in the hspA promoter and in the promoter of the photoinducible genes carB and carRA; and these are candidates as binding sites for light-regulated transcription factors. Blue light can increase transcription of the hspA gene 10-fold, with a threshold of 1 J/m2. The threshold for hspA photoactivation is 10(4) times higher than the thresholds for blue-light regulation of sporangiophore development and photocarotenogenesis, which suggests that there are differences in the photosensory systems for gene photoactivation and mycelial photoresponses. A heat-shock of 30 min at 34 degrees C or 42 degrees C increased hspA gene activity 160-fold. The differences in maximum hspA gene transcription by blue light and heatshock suggest the presence of different regulatory mechanisms.

Involvement of G proteins in the mycelial photoresponses of Phycomyces.

Many responses of the zygomycete fungus Phycomyces blakesleeanus are mediated by blue light, e.g. the stimulation of beta-carotene synthesis (photocarotenogenesis) and the formation of fruiting bodies (photomorphogenesis). Even though both responses have been described in detail genetically and biophysically, the underlying molecular events remain unknown. Applying a pharmacological approach in developing mycelia, we investigated the possible involvement of heterotrimeric G proteins in the blue-light transduction chains of both responses. G protein agonists (guanosine triphosphate analogues, cholera toxin, pertussis toxin) mimicked in darkness the effect of blue light for both responses, except for cholera toxin, which was ineffective in increasing the beta-carotene content of dark-grown mycelia. Experiments combining the two toxins indicated that photocarotenogenesis could involve an inhibitory G protein (Gi) type, whereas photomorphogenesis may depend on a transducin (Gt type)-like heterotrimer. The determination of the carB (phytoene dehydrogenase) and chs1 (chitin synthase 1) gene expression under various conditions of exogenous challenge supports the G protein participation. The fluctuations of the time course measurements of the carB and chs1 transcripts are discussed.

Light-induced fluorescence changes in Phycomyces: evidence for blue light-receptor associated flavo-semiquinones.

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH*), and the flavo-hydroquinone (FlH2) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH2, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.

Tropisms in Phycomyces: sine law for gravitropism, exponential law for photogravitropic equilibrium.

Sporangiophores of Phycomyces blakesleeanus that are gravitropically stimulated by inclining them relative to the earth's gravitational vector obey the sine law for inclination angles between 0 degrees and 150 degrees. The quantitative relation between gravitropism and phototropism was analyzed for sporangiophores that were kept in balance between opposing gravitational and phototropic stimuli. The gravitropism of inclined sporangiophores was compensated with unilateral light impinging at right angles relative to the axis of the sporangiophore. The fluence rate of unilateral blue light (466 nm) that was required to counteract the negative gravitropism increased exponentially with the sine of the inclination angle of the sporangiophore. The establishment of photogravitropic equilibrium during continuous unilateral irradiation is thus determined by two different laws: the well-known sine law for gravitropism and a novel exponential law of phototropism described in this work. Furthermore, the specific form of the exponential relationship depends on the presence of statoliths (vacuolar protein crystals) and on wavelength.

Role of nitric oxide synthase in the light-induced development of sporangiophores in Phycomyces blakesleeanus.

Blue light controls the development of sporangiophores in the zygomycete Phycomyces blakesleeanus Burgeff. Light represses the production of microsporangiophores and enhances the development of macrosporangiophores. Inhibition of the biosynthesis of tetrahydrobiopterin, a cofactor of NO synthase, inhibits this photomorphogenesis. Light induces production of citrulline from arginine in the mycelium and in sporangiophores. The citrulline-forming activity is dependent on NADPH, independent of calcium, and inhibited by NO synthase inhibitors. It is reduced in tetrahydrobiopterin-depleted mycelium. Light induces emission of NO from the developing fungus in the same order of magnitude as citrulline formation from arginine. The NO donor sodium nitroprusside can replace the light effect on sporangiophore development, and inhibitors of NO synthase repress it. We suggest that a fungal NO synthase is involved in sporangiophore development and propose its participation in light signaling.

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

To elucidate the graviperception of the unicellular fungus, Phycomyces blakesleeanus, 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 degrees 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 micrometers 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 degrees-50 degrees upward. In stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 micrometers 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.

Reception of far-ultraviolet light in Phycomyces: antagonistic interaction with blue and red light.

Phototropism experiments were done with sporangiophores of the fungus Phycomyces blakesleeanus to characterize the interaction between far-UV, blue and red light. Far-UV light elicits negative phototropism (bending away from the light source) while blue light elicits positive phototropism (bending toward the light source). In contrast, red light above 600 nm is phototropically inert. Phototropism was analyzed with light regimens of bilateral or unilateral irradiation with far-UV and blue light. Under bilateral irradiation, in which the two light sources were facing each other, blue light partially inhibited the far-UV-elicited phototropism. A fluence-response curve for this inhibition showed that blue light was maximally effective at fluence rates which exceeded 3 to 57 times that of the far-UV. Tonic red light, which was given from above, abolished to a large extent the antagonistic action of blue light. With a regimen of unilateral irradiation, i.e. when far-UV and blue light were given from the same side, a phototropic balance could be achieved with approximately equal fluence rates of blue and UV light. Above or below this critical balance point the bending was either negative or positive. In this setup the effect of tonic red light was complex. First, it caused an enhancement of the positive or negative bending, and second, it caused at some fluence rates a sign reversal from positive to negative phototropism. The balance point itself was only marginally affected. The data cannot be explained on the basis of a single photoreceptor and support the previous notion of separate far-UV and blue-light receptors. The antagonism between these two receptors probably occurs on the level of a red-light-absorbing receptor intermediate.

Mutants of phycomyces with decreased gallic acid content.

Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycete Phycomyces 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 isolate hba mutants 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 the hba mutant only slightly better than the wild type.

Lack of protection by carotenes against gamma-radiation damage in Phycomyces.

Carotenes could protect cells from radiation damage by chemically quenching the free radicals and the activated chemical species originated by the exposure. We tested this hypothesis with strains of the zygomycete Phycomyces blakesleeanus that contained different carotenes (phytoene, lycopene, beta-carotene) or different concentrations of beta-carotene. Pairs of strains were cultured together, exposed to a maximum of 73 Gy gamma-radiation from a Co source, and allowed to recover and grow further together on limited resources. Irradiation did not affect the relative abundance of each strain in the resulting spore crop. Thus, carotenes did not protect the fungal cells against gamma-radiation and did not influence their recovery from damage caused by the exposure.