Vegetative regeneration on sexual organs inPhycomyces 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.     

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.     

The sensitive period for light and temperature regulation of sporangiophore development inPhycomyces

Light and temperature markedly influence sporangiophore development inPhycomyces blakesleeanus. Under normal conditions in the dark, low temperature drastically stimulates the production of dwarf sporangiophores (microphorogenesis) and inhibits that of giant sporangiophores (macrophorogenesis). These effects of low temperature could still be observed if applied only for a short period before sporangiophore initiation. Continuous white illumination strongly inhibits microphorogenesis and slightly stimulates macrophorogenesis. Short exposures to white light noticeably inhibit microphorogenesis and stimulate macrophorogenesis when given to mycelia grown for between 90 and 160 h at 14° C or 150 h or more at 10° C. These results indicate the existence in the mycelium of developmental stages for the regulation of sporangiophorogenesis by environmental signals.     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

Growth-rate adaptation of Phycomyces sporangiophores to partial depletion of oxygen

The growth rate of Phycomyces blakesleeanus sporangiophores was found to be very sensitive to sudden changes in the oxygen concentration. A change from 20% to 15% oxygen elicits a transient decrease in the growth rate which returns to normal 10 min after altering the concentration. After a step change to 10% oxygen, the growth rate shows two minima at 6–8 and 30–35 min and it reaches about 80% of its original value 50 min after this change. A threshold curve for this negative growth response shows that sporangiophores begin to sense a decrease in the oxygen concentration from 20% to 17%. Seven phototropically abnormal mutants with defects in the genes madA to madG were tested for the oxygen response. Two strains, C149madD120 and C316madF48, were found to have recoveries different from those of the wild type after step changes from 20% to 10% oxygen.     

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 m. 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.Abbreviations spph(s) sporangiophore(s) - wt(s) wild type(s)     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.     

Phycomyces: Phototropism and light-growth response to pulse stimuli

The relationship between phototropism and the light-growth response of Phycomyces blakesleeanus (Burgeff) sporangiophores was investigated. After dark adaptation, stage-IVb sporangiophores were exposed to short pulses of unilateral light at 450 nm wavelength. The sporangiophores show a complex reaction to pulses of 30 s duration: maximal positive bending at 3·10^-4 and 10^-1 J m^-2, but negative bending at 30 J m^-2. The fluence dependence for the light-growth response also is complex, but in a different way than for phototropism; the first maximal response occurs at 1.8·10^-3 J m^-2 with a lesser maximum at 30 J m^-2. A hypertropic mutant, L85 (madH), lacks the negative phototropism at 30 J m^-2 but gives results otherwise similar to the wild type. The reciprocity rule was tested for several combinations of fluence rates and pulse durations that ranged from 1 ms to 30 s. Near the threshold fluence (3·10^-5 J m^-2), both responses increase for pulse durations below 67 ms and both have an optimum at 2 ms. At a fluence of 2.4·10^-3 J m^-2, both responses decrease for pulse durations below 67 ms. The hypertropic mutant (madH), investigated for low fluence only, gave similar results. In both strains, the time courses for phototropism and light-growth response, after single short pulses of various durations, show no clear correlation. These results imply that phototropism cannot be caused by linear superposition of localized light-growth responses; rather, they point to redistribution of growth substances as the cause of phototropism.     

Electrophoretic analysis of proteins fromPhycomyces mutants with abnormal tropisms

Certain phototropism mutants ofPhycomyces blakesleeanus show defective bending responses (tropisms) to stimuli besides light, such as gravity, wind, and barriers. These so-called stiff mutants are affected in four genes (madD tomadG). Using two-dimensional gel electrophoresis, we have analyzed polypeptides from microsomal and soluble fractions obtained from the wild type, four single mutants, and six double mutants affected in all pairwise combinations of the four genes. Consistent differences in spot patterns formadE andmadF mutants were found in microsomal fractions but not in soluble fractions. InmadE mutants, two spots designated E1 (52 kDa,pI 6.65) and E2 (50 kDa,pI 6.65) were altered. E1 appeared denser in the wild type than in themadE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control bymadE, perhaps involving posttranslational modification. A protein spot, F1 (53 kDa,pI 6.1), was present on the wild-type gels but absent from all gels formadF mutants. The F1 polypeptide probably represents themadF gene product.This work was supported by research grants to E.D.L. from the National Science Foundation (DMB-8316458 and DMB-8704602) and an equipment grant to C.H.T. from the Syracuse University Senate Research Committee.     

Gravity susception by buoyancy: floating lipid globules in sporangiophores of<Emphasis Type="Italic"> Phycomyces</Emphasis>

To elucidate the mechanisms of gravity susception that operate in the sporangiophore of Phycomyces blakesleeanus, we characterized the function and topography of a large apical complex of lipid globules. Stage-1 sporangiophores (without sporangium) possess a roughly spherical complex of 100–200 large lipid globules whose center is localized 110 m below the apex. The complex of lipid globules (CLG) is rather stable and is kept in place by positioning forces that resist centrifugal accelerations of up to 150 g. The lipid globules possess an average diameter of 2 to 2.5 m and a density of 0.791 g cm^–3, which is below that of typical plant oleosomes. The potential energy which is generated by the buoyancy of a CLG of 100 globules is in the order of 10^-17 to 10^-16 J, which is 4 to 5 orders of magnitude above thermal noise. The formation of lipid globules can be supressed by raising stage-1 sporangiophores for 24 hs at 5°C. Sporangiophores with a reduced number of lipid globules display gravitropic bending angles that are 3 to 4 times smaller than those of sporangiophores with the normal number of lipid globules. The results suggest that the lipid globules function as gravisusceptors of Phycomyces and that buoyancy is the physical principle for their mode of action. The globules contain -carotene and two distinct fluorescing pigments that are, however, dispensible for graviperception.Abbreviations CLG complex of lipid globules     

A genetic map of Phycomyces blakesleeanus

Summary Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.     

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affected in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent components of the photoreceptor complex responsible for phototropism in Phycomyces.This work was supported in part by an equipment grant to JAP from the Syracuse University Senate Research Committee, research grants to EDL from the National Science Foundation (PCM-8003915 and DMB-8316458), and a fellowship to EDL from the Alfred P. Sloan Foundation.     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces 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.     

A comparison of cell-wall-yielding properties for two developmental stages of Phycomyces sporangiophores

The yielding properties of the cell wall, irreversible wall extensibility (m) and yield threshold (Y), are determined for stage I sporangiophores of Phycomyces blakesleeanus from in-vivo creep experiments, and compared to the values of m and Y previously determined for stage IVb sporangiophores using the same pressureprobe method (Ortega et al., 1989, Biophys. J. 56, 465). In either stage the sporangiophore enlarges (grows) predominately in length, in a specific region termed the growing zone, but the growth rates of stage I (5–20 urn · min^–1) are smaller than those of stage IVb (30–70 m · min^–1). The results demonstrate that this difference in growth rate is the consequence of a smaller magnitude of m for stage I sporangiophores; the obtained values of P (turgor pressure), Y, and P-Y (effective turgor for irreversible wall extension) for stage I sporangiophores are slightly larger than those of stage IVb sporangiophores. Also, it is shown that the magnitude of m for the stage I sporangiophore is regulated by altering the length of the growing zone, L_g. A relationship between m and L_g is obtained which can account for the difference between values of m determined for stage I and stage IVb sporangiophores. Finally, it is shown that similar changes in the magnitude of m and (which have been used interchangeably in the literature as a measure of irreversible wall extensibility) may not always represent the same changes in the cell-wall properties.Abbreviations and Symbols L length - L_g length of growing zone - m irreversible wall extensibility - P turgor pressure - Y yield threshold - (P-Y) effective turgor for irreversible wall extension - relative irreversible wall extensibility - _g relative irreversible wall extensibility of the growing zone (m/L_g) This work was supported by National Science Foundation grant DCB-8801717 to J.K.E. Ortega.     

Photomorphogenesis inPhycomyces: Dependence on environmental conditions

The production of two kinds of vegetative reproductive structures, microphores and macrophores, byPhycomyces blakesleeanus Bgff. depends on plating density, ventilation, asparagine supply, and illumination. Quantitative determinations of these variables lead us to propose a new experimental system for developmental photobiology: standard plastic Petri plates containing 25 ml minimal medium are inoculated with 10⁵ viable, heat-activated spores and incubated, unpiled and unsealed, at 22° C. After 4 d microphores are counted and macrophores are weighed. Both microphorogenesis and macrophorogenesis are governed by light. Photosensitivity is a developmental phenomenon which occurs 32 to 68 h after inoculation, just before the beginning of vegetative reproduction in the dark controls. The maximum photosensitivity occurs 48 h after inoculation.     

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.     

Effects of chemical components and nitrogen sources on zygospore development inPhycomyces blakesleeanus

We examined the effects of chemical components and nitrogen sources on zygospore development, using 62 different ingredients based on Sutter's synthetic medium Sl, which has been widely used for studies of sexual physiology inPhycomyces. An increase of inorganic microelements such as ZnSO₄, NaMoO₄ and CaCl₂ promoted an increase in the number of zygospores per unit area. Glutamate (Glu) contained in Sl as the sole nitrogen source was indispensable for sexual development, and replacement of Glu with NH₄ ⁺ (Am) strongly inhibited it, mainly because of growth inhibition. However, zygospore production was enhanced 1.8-fold by equivalent amounts of both Glu and Am as compared with Glu alone. A newly developed medium, mSl+Am, enriched with Am and the above-mentioned effective microelements doubled the number of zygospores formed per unit area (density), compared with Sutter's original Sl, and increased both the density and the weight (volume) of zygospores, 1.6- and 2-fold, respectively, compared with potato-dextrose-agar medium enriched with yeast extract and casitone (PDAYC). Sexual stimulation by mSl+Am was also observed in the mating of a pair of β-carotene-deficient mutants. Methionine sulfoxime, an inhibitor of glutamine synthetase, strongly inhibited the progress of mating without significant growth inhibition. The first and the second authors contributed equally to this work.     

The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 ± 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (di-nitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N,N-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the upper and lower sides of gravistimulated maize root tips which is necessary for gravitropism.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - FC fusicoccin - Mes 2-(N-morpholino)ethanesulfonic acid The authors thank Phyllis Woolwine for drawing Fig. 1, Dr. Sarbjit Virk for assistance with total calcium measurements, Dr. Paul Sampson for statistical advice, and Michael Newton for developing the EM algorithm to analyze the time-series data. This work was supported by NASA grant NAGW-1394 and by a NASA Research Associateship to T.B. through NASA grant NAGW-70.