Tamotsu Ootaki

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

Abstract. To elucidate the graviperception of the unicellular fungus, Phycomycesblakesleeanus, 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° 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 μm 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°–50° upward. In stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 μm 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.

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses of Phycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.

Parameters governing gravitropic response of sporangiophores in Phycomyces blakesleeanus

The sporangiophores (spphs) of the fungusPhycomyces blakesleeanus bend upward in a negative gravitropic response when placed in a horizontal position in the dark. The spphs of a hypergravitropic mutant showed higher bending rate and shorter latency period than those of the wild type. In both strains, spphs of smaller diameter had higher bending rates. No significant differences were found between the wild type and the mutant and between the thin spphs and the spphs of standard diameter in respect to their elongation rates. Phototropic rate was also the same between the wild type and the mutant. Parameters influencing the gravitropic response such as diameter of the spph, absolute elongation rate, and ratio of differential growth between the upper and the lower sides of the extension zone of spph were investigated to elucidate the kinetics of bending in the mutant. The results demonstrate that the rapid gravitropic response in the mutant is due to its higher (about 5–6 times) differential-growth rate compared with the wild type.

The determination of the maximal bending angle inPhycomyces sporangiophores

Abstract The maximal phototropic bending angle of the Phycomyces sporangiophore (spph) was always smaller than 90° from the vertical. Experimental results, using a clinostat apparatus and both a gravitropic mutant and the wild-type spphs placed vertically or horizontally and illuminated from different directions, revealed that this angle resulted from a balance between a negative gravitropism and a phototropism whose direction (positive or negative) and magnitude depended on the bending angle of the spph, because of the involvement of the optical properties of the spph, probably the ratio of the maximal light-fluence rate between the proximal side ( I P,max ) and the distal side ( I D,max ) of the spph. Shadowing of the extension zone by the sporangium was estimated to be complete only when the bending angle was larger than 86.4° from the vertical.

Roles of extracellular fibrils connecting progametangia in mating of Phycomyces blakesleeanus

The mating progametangial cells of the opposite mating types of Phycomyces are connected by extracellular fibrils. These fibrils were poorly constructed when Phycomyces was mated at high temperature (25 °C), when many progametangia were abnormally twisted or deformed and ceased further development. Surgical destruction of the fibrils also often resulted in twisting or deformation of progametangia and in cessation of the mating process. These results imply that the fibrils may play a role to tie two progametangial cells and to maintain their normal three-dimensional configuration, which is essential for further mating development.

Diameter and amount of β-carotene determine the maximal phototropic bending angle ofPhycomyces sporangiophores

Wild-type sporangiophores at stage IVb (final developmental stage after sporangium formation) ofPhycomyces show a pronounced positive phototropism to unilateral white light. We found that the maximal bending angle was larger in thin sporangiophores than in thick ones, and larger in the sporangiophores containing a small amount of β-carotene than in those containing a large amount of it. These phenomena probably occur because of the increase in length of intracellular light path or in the intracellular light-attenuation coefficient, as supported theoretically.

Developmental stage-dependent response of Pilobolus 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.

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.

Vegetative regeneration on sexual organs in Phycomyces blakesleeanus

Phycomyces blakesleeanus produced an abundance of sexual organs when two mating types met on solid medium, but only about 14.7% of the sexual organs developed to the final stages. On the sexual organs showing arrested development, vegetative hyphae or dwarf sporangiophores (microphores) often regenerated. This vegetative regeneration was accelerated when the paired and looped progametangia were isolated from mycelia, when the counterparts of the progametangial cells constructing the loop were surgically incised, and whenPhycomyces was mated at high temperature (25–27°C). A leaky-carotenogenic mutant, whose sexual reaction was imperfect and arrested at an intermediate stage even when mated with the wild type, also regenerated hyphae with a high frequency on these arrested intermediate organs. The vegetative regeneration seems to result from interruption of a cell-to-cell recognition system between cells of different mating type, which is believed to be essential for the mating process of this fungus in addition to the pheromonal actions.