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.     

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)     

Photoinduced accumulation of carotene in Phycomyces

Blue light stimulates the accumulation of beta-carotene (photocarotenogenesis) in the fungus Phycomyces blakesleeanus. To be effective, light must be given during a defined period of development, which immediately precedes the cessation of mycelial growth and the depletion of the glucose supply. The competence periods for photocarotenogenesis and photomorphogenesis in Phycomyces are the same when they are tested in the same mycelium. Photocarotenogenesis exhibits a two-step dependence on exposure, as if it resulted from the additon of two separate components with different thresholds and amplitudes. The low-exposure component produces a small beta-carotene accumulation, in comparison with that of dark-grown mycelia. The high-exposure component has a threshold of about 100 J· m^–2 blue light and produces a large beta-carotene accumulation, which is not saturated at 2·10⁶ J·m^–2. Exposure-response curves were obtained at 12 wavelengths from 347 to 567 nm. The action spectra of the two components share general similarities with one another and with those of other Phycomyces photoresponses. The small, but significant differences in the action spectra of the two components imply that the respective photosystems are not identical. Light stimulates the carotene pathway in the carB mutants, which contain the colourless precursor phytoene, but not beta-carotene. Carotenogenesis is not photoinducible in carA mutants, independently of their carotene content. This and other observations on various car mutants indicate that light prevents the normal inhibition of the pathway by the carA and carS gene products. The chromophore(s) for photocarotenogenesis are presumably flavins, and not carotenes.We thank Dr. A. Palit, C. Chmielewicz and D. Durant (same address as E.D.L.) and L.M. Corrochano, A. Fernández Estefane, and J. Córdoba López (same address as E.R.B.) for their help. This work was supported by grants from Comisión Asesora para Investigación Científica y Técnica and Comisión Interministerial para Ciencia y Tecnología to E.C.O.; from the National Institutes of Health and the National Science Foundation to E.D.L.; and from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation to E.C.O. and E.D.L.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Subcellular localization of calcium in sporangiophores of Phycomyces blackesleeanus

The in situ localization of Ca²⁺ in stage I sporangiophores of the fungus Phycomyces blakesleeanus was achieved with the potassium pyroantimonate technique. Precipitates of calcium-antimonate were present in mitochondria, vacuoles, endoplasmic reticulum and adjacent cytoplasm, Golgi-like bodies, and nuclei but not cell walls. Material treated with the calcium chelator EGTA lacked these precipitates. The preferential localization of Ca²⁺ in mitochondria, endoplasmic reticulum and vacuoles suggests that these organelles modulate the level of this cation in sporangiophores of P. blakesleeanus.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N, tetraacetic acid     

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.     

Analysis of microsomal flavoproteins from Phycomyces sporangiophores: Candidates for the blue-light photoreceptor

To help identify components of the blue-light photoreceptor system for phototropism in Phycomyces blakesleeanus Bgff., proteins from a microsomal fraction obtained from synchronous sporangiophores were studied. By two-dimensional gel electrophoresis, two proteins (FP₁, FP₂) with covalently bound flavins were found. FP₁ has a molecular weight of 71 000 and an isoelectric point of 6.6; FP₂ has a molecular weight of 59 000 and an isoelectric point of 6.5. These flavoproteins were purified by column chromatography and gel filtration while assaying for flavins by fluorescence. The relative concentrations of FP₁ and FP₂ were affected by light applied during growth. These flavoproteins are likely components of the blue-light photoreceptor complex mediating phototropism in Phycomyces.Abbreviations 10 k pellet 10 000-g pellet - 100 k pellet 100 000-g pellet - FP₁, FP₂ proteins with covalently bound flavins having molecular weights of 71 000 and 59 000 and isoelectric points of 6.6 and 6.5, respectively     

Action spectra of the light-growth response of Phycomyces

The light-growth response of Phycomyces blakesleeanus (Burgeff) is a transient change in elongation rate of the sporangiophore caused by a change in light intensity. Previous investigators have found that the light-growth response has many features in common with phototropism; the major difference is that only the light-growth response is adaptive. In order to better understand the light-growth response and its relationship to phototropism, we have developed a novel experimental protocol for determining light-growth-response action spectra and have examined the effect of the reference wavelength and intensity on the shape of the action spectrum. The null-point action spectrum obtained with broadband-blue reference light has a small peak near 400 nm, a flat region from 430 nm to 470 nm, and an approximately linear decline in the logarithm of relative effectiveness above 490 nm. The shape of the action spectrum is different when 450-nm reference light is used, as has been shown previously for the phototropic-balance action spectrum. However, the action spectrum of the light-growth response differs from that for phototropic balance, even when the same reference light (450 nm) is used. Moreover, for the light-growth response, the relative effectiveness of 383-nm light decreases as the intensity of the 450-nm reference light increases; this trend is the opposite of that previously found for phototropic balance. The dependence of the lightgrowth-response action spectrum on the reference wavelength, its difference from the phototropic-balance action spectrum, and the reference-intensity dependence of the relative effectiveness at 383 nm may be attributable to dichroic effects of the oriented photoreceptor(s), and to transduction processes that are unique to the light-growth response.I dedicated to Masaki Furuya on the occasion of his 65th birthdayThis work was supported by a grant from the National Institutes of Health (GM29707) to E.D. Lipson. Anuradha Palit, Promod Pratap, and Benjamin Horwitz participated in the early phases of this work. We thank Leonid Fukshansky and Benjamin Horwitz for helpful discussions, David Durant for computer programming, and Steven Block for providing us with a C-language program of Reinsch's procedure for cubic spline interpolation. One of us (R.S.) gratefully acknowledges a junior faculty fellowship leave from the Department of Physics at Yale University.     

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.     

Action spectra of the light-growth response in three behavioral mutants of Phycomyces

Null-point action spectra of the light-growth response were measured for three mutants of Phycomyces blakesleeanus (Burgeff) and compared with the action spectrum of the wild type (WT). The action spectrum for L150, a recently isolated night-blind mutant, differs from the WT spectrum. The L150 action spectrum has a depression near 450 nm and small alterations in its long-wavelength cutoff, the same spectral regions where its photogravitropism action spectrum is altered. This indicates that the affected gene product influences both phototropism and the light-growth response. For L85, a hypertropic (madH) mutant, the light-growth-response action spectrum is very similar to that of WT even though the photogravitropism action spectrum of L85 has been shown previously to be altered in the near-UV region. The affected gene product in this mutant appears to affect phototropic transduction but not light-growth-response transduction. The action spectrum of C110, a stiff (madE) mutant, differs significantly from the WT spectrum near 500 nm, the same spectral region where sporangiophores of madE mutants have been shown to have small alterations in second-derivative absorption spectra. This indicates that the madE gene product may be physically associated with a photoreceptor complex, as predicted by system-analysis studies.Abbreviations SE standard error of the mean - UV ultraviolet light - Wt wild type I dedicated to Masaki Furuya on the occasion of his 65th birthdayWe thank H. Reiner Schaefer for performing some of the experiments and for help in data analysis, David Durant for computer programming, and Benjamin Horwitz for helpful discussions. This work was supported by a grant from the National Institutes of Health (GM29707) to E.D. Lipson.     

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     

Detoxification of N-(phosphonoacetyl)-L-aspartate by carrot cells in suspension culture

Phototropic reversal of Phycomyces sporangiophores can be elicited by a change to darkness during steady-state phototropism. The reversal lasts 25–30 min under these conditions. Control experiments show that the reversal is not caused by gravitropism. Tropic reversal is also elicited by the removal of a barrier during an avoidance response, showing that the reversal occurs at the output of the sensory transduction chain.     

Blue-light-induced shift of the phototropic fluence-response curve in Pilobolus sporangiophores

The effects of preirradiation with blue light on the shift of the fluence-response curve for the first and the second positive curvatures were examined in Pilobolus crystallinus (Wiggers) Tode sporangiophores. A 1-min preirradiation with blue light at 47 or 960 nmol·m^-2 lowered the fluence-response curve for the first positive curvature and shifted the peak to a higher fluence. The fluence-response curve was shifted back to a lower fluence when a dark period was inserted between the preirradiation and the curvature-inducing light. This shift back to lower fluence was biphasic when the fluence was high (960 nmol · m^-2), indicating the participation of two components in the phototropic reaction for the first positive curvature.The fluence-response curve for the second positive curvature did not seem to be shifted to a higher fluence region when fluence was varied by varying exposure time. However, the fluence-response curve obtained by varying the fluence rate of a 20-min irradiation period indicated that the second positive curvature was also shifted to a higher-fluence region by preirradiation with blue light. A small shoulder appeared on the fluence-response curve when preirradiation at a high fluence rate was given.Abbreviations BL blue light - CIL curvature-inducing light     

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.     

Parameters governing gravitropic response of sporangiophores inPhycomyces 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.     

End-product regulation of carotenogenesis in Phycomyces

Wild-type Phycomyces blakesleeanus synthesizes the yellow pigment, beta-carotene. Colour mutants exhibit various alterations in the biosynthesis of beta-carotene or in its regulation. The presence of certain chemicals in the medium stimulates carotenogenesis in the wild type. We attribute different mechanisms of action to agents which stimulate or fail to stimulate different sets of mutants; this is the case of retinol and dimethyl phthalate. Dimethyl phthalate and veratrol are active on the same mutants, and therefore are likely to act in the same way. The main regulation of carotenogenesis, end-product inhibition, does not operate in the mutants of certain genes; these mutants are indifferent to retinol. By using a collection of retinoids we conclude that their action depends on their structural similarity to a part of the beta-carotene molecule. From these and other observations we propose that end-product inhibition of the pathway is mediated by a complex of beta-carotene and two gene products and that the retinoids compete with beta-carotene and prevent end-product inhibition.Deceased     

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.Abbreviations CW clockwise - CCW counterclockwise — both as viewed from above     

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.This work was carried out under the Joint Research Program of the Institute of Genetic Ecology, Tohoku University, Japan (892006). The authors please to thank Kaori Koga and Hiroko Kikuchi for their helpful assistance.     

Ethylene is involved in the autochemotropism of Phycomyces

The sporangiophore of the fungus Phycomyces blakesleeanus has the property of growing away from a barrier which is few mm from the growing zone of the sporangiophore (avoidance or autochemotropic response). A model has been published (Cohen, R.J., Jan, N.Y., Matricon, J., Delbrück, M.: J. Gen. Physiol. 66, 67–95 (1975)). To explain the avoidance response which postulates that the sporangiophore emits and readsorbs a volatile growth-promoting effector (gas X) and that the barrier modifies the effector distribution by acting as an aerodynamic obstacle, causing a higher concentration of gas X on the side of the sporangiophore closer to the barrier. From this model we deduced three properties of the gas X. Of the several gases tested (N₂, CO₂, CH₄, C₂H₂, C₂H₄, C₂H₆) only ethylene (C₂H₄) had all these three properties, a finding which suggests that it has a role in the avoidance response (autochemotropism).Abbreviation Spph Sporangiophore     

Trehalose metabolism in dormant and activated spores of Phycomyces blakesleeanus Burgeff

Evidence is obtained for the existence of two different localizations of trehalase (,-trehalose glucohydrolase, EC 3.2.1.28) in Phycomyces spores: one inside the cell, and one in the periplasmic region. The latter enzyme is sensitive to 0.1 mol l^-1 HCl treatment and its activity can be regulated by external pH changes. The periplasmic form of the enzyme is involved in the metabolism of added labelled trehalose. This sugar is hydrolyzed externally to glucose which is found mainly in the incubation medium and which is partly absorbed by the spores. During incubation trehalose leaks out from both dormant and activated spores and is subsequently hydrolyzed to glucose. The intracellular trehalase is probably involved in the breakdown of endogenous trehalose in spores. After heat activation the hydrolysis of endogenous trehalose is stimulated even without an important increase in activity of intracellular trehalase. Additional treatments which break dormancy of spores without a significant activation of trehalase are the following: heating of HCl-treated spores and treatment of spores with reducing substances (e.g. Na₂S₂O₄ and NaHSO₃).