Statoliths in Phycomyces: characterization of octahedral protein crystals.

To identify the molecular mechanisms of gravitropism in the fungus Phycomyces blakesleeanus we determined several biochemical and physical parameters of paracrystalline protein bodies, so-called octahedral crystals. The crystals, which are present throughout the central vacuoles of the sporangiophore, function as statoliths (Schimek et al., 1999a,b). They possess an average volume of 9.96 microm(3) and a specific mass of 1.26 g cm(-3). SDS-PAGE of purified crystals shows three major proteins with relative molecular masses of 16, 46.5, and 55 kDa. These proteins are absent in gravitropism mutants which lack the crystals. Phototropism mutants (genotype mad) which are graviresponsive (class 1) and those which are defective in gravitropism (class 2) contain the crystals and the three associated proteins. Absorption spectra of isolated crystals and in situ absorption spectra of growing zones indicate the presence of chromophores, probably oxidized and reduced flavins. The flavin nature of the chromophores is also indicated by their fluorescence properties. It appears likely that the chromophores represent an essential part of the statoliths and thus the gravitropic transduction chain.

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.

Protein crystals in Phycomyces sporangiophores are involved in graviperception.

The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.

Chitin synthase-encoding gene(s) of the Zygomycete fungus Phycomyces blakesleeanus.

Using polymerase chain reaction (PCR) primers to two highly conserved sequences within fungal chitin synthase (CHS)-encoding genes, an approximately 750-bp DNA fragment was amplified from genomic DNA of Phycomyces blakesleeanus. The amino acid sequence deduced from the nucleotide sequence of this fragment best matches the motifs found in class-II CHS. The fragment includes an approximately 160-bp region that likely is an intron. Southern hybridization of restriction enzyme-digested genomic DNA, using the PCR-amplified DNA as the probe, suggests that P. blakesleeanus contains additional CHS-encoding genes (CHS). To our knowledge, this is the first report on the detection of a CHS gene in a Zygomycete fungus. These studies represent a major step toward exploring the molecular mechanisms of CHS regulation in Phycomyces. The prospects are exciting, since CHS is implicated to play a central role in the sensory responses of P. blakesleeanus involving growth modulations.

Negative phototropism in the piloboloid mutants of Phycomyces blakesleeanus Bgff.

The sporangiophore (spph) of a piloboloid mutant, genotype pil, of Phycomyces ceases elongation and expands radially in the growth zone shortly after reaching the developmental stage IV b. The pil spph is always negatively phototropic to unilateral visible light when its diameter exceeds 210 µm. Photoinduction of spph initiation, light-growth response, threshold of light energy fluence rate for the negative phototropism, avoidance and gravitropism in the pil mutant are all normal. In liquid paraffin, the pil spph shows negative phototropism as does the wild-type spph. Genetic analyses indicate that the negative phototropism of the pil mutant is governed by the phenotypic characteristics of pil but not by specific gene(s) responsible for negative phototropism. These facts imply that the reverse phototropism of the pil mutant results from a loss of the convergent lens effect of the cell because of the increase in cell diameter.

Spiral growth in the radially-expanding piloboloid mutants ofPhycomyces blakesleeanus.

The growth zone of the sporangiophore of a piloboloid mutant,pil, ofPhycomyces expands radially at an increased rate until the growth zone becomes nearly spherical, in sharp contrast to that of the wild-type sporangiophore which exhibits longitudinal elongation only and is conical. The rotation of thepil sporangiophore reverses its direction from clockwise (CW) to counterclockwise (CCW) during the period of increased radial expansion, and the CCW rotation continues as long as does the radial expansion. The direction of rotation and the time of reversal are correlated with the relative rates of cell-wall expansion in the longitudinal and transverse directions. The CCW rotation of the sporangiophore of this mutant can be explained by the behavior of the microfibrils, as previously proposed to explain the rotation of the wild-type sporangiophore.

An unstable nuclear gene in phycomyces.

A gentic instability in Phycomyces is described that appears to be associated with a single nuclear gene, dar. The wild type is able to take up riboflavin and its toxic analogue, deaza-riboflavin, from nanomolar concentrations in the medium. The mutants are unable to take up riboflavin and are resistant to deaza-riboflavin. Forward and reverse mutation rates are estimated to be 4 x 10(-5) and 2 x 10(-3) per nuclear division. Independently arisen dar mutants do not complement in heterokaryons. The mutant alleles are almost completely recessive. The phenotype of spores is not determined cell-autonomously, but is strongly influenced by the allele ratio among the nuclei in the sporangium of origin.

Octahedral crystals in Phycomyces. II.

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the beta-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265-285 nm, 350-380 nm, and 450-470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".