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     

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.     

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)     

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.     

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.     

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.     

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.     

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.     

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     

On the relation between photo- and gravitropically induced spatial memory in maize coleoptiles

The interaction of photo- and gravitropic stimulation was studied by analysing the curvature of maize (Zea mays L.) coleoptiles subjected to rotation on horizontal clinostats. Gravitropic curvature in different directions with respect to the stimulation plane was found to be transient. This instability was caused by an increasing deviation of response direction from the stimulation plane towards the caryopsis. The bending angle as such, however, increased steadily. This reorientation of the gravitropic response towards the caryopsis is thought to be caused by the clinostat-elicited nastic curvature found in maize coleoptiles. In contrast, the response to phototropic stimulation was stable, in both, orientation and curving. Although stimulation by gravity was not capable of inducing a stable tropistic response, it could inhibit the response to opposing phototropic stimulation, if the counterstimulation was given more than 90 min after the onset of gravistimulation. For shorter time intervals the influence of the phototropic stimulus obscured the response to the first, gravitropic stimulation. For time intervals exceeding 90 min, however, the phototropic effects disappeared and the response was identical to that for gravity stimulation alone. This gravity-induced inhibition of the phototropic response was confined to the plane of gravity stimulation, because a phototropic stimulation in the perpendicular direction remained unaffected, irrespective of the time interval between the stimulations. This concerned not only the stable phototropic curving, but also the capacity of the phototropic induction to elicit a stable directional memory as described earlier (P. Nick and F. Schäfer, 1988b, Planta 175, 380–388). This was tested by a second bluelight pulse opposing the first. It is suggested that gravity, too, can induce a directional memory differing from the blue-light elicited memory. The mechanisms mediating gravi- and phototropic directional memories are thought to branch off the respective tropistic signal chains at a stage where photo- and gravitropic transduction are still separate.This work was supported by the Deutsche Forschungsgemeinschaft and a grant of the Studienstiftung des Deutschen Volkes to P. Nick.     

The Phycomyces lens: measurement of the sporangiophore intensity profile using a fiber optic microprobe

A fiber optic microprobe, 5.5 m in diameter, was used as a detector to measure the light intensity profile at the distal cell surface of Phycomyces blakesleeanus (Burgeff) sporangiophores that were irradiated unilaterally by a collimated xenon source. The light intensity at a fixed location of the cell surface showed large random variations over time which were probably the result of optical effects of particles being carried past the probe by cytoplasmic streaming. The intensity profile, formed around the distal periphery of the cell by the lens action of the sporangiophore, was determined from intensity measurements made while the probe was held fixed and the incident beam direction was varied in angle of azimuth. The resulting profile consisted of two steeply rising sides enclosing a central plateau or shallow well which ranged in fluence rate from 1.6 to 2.2 times that of the incident beam. These experimental findings differ from theoretical modeling where much greater contrast between the sides and central portion of the lens profile was predicted. These results also indicate that the mechanism of phototropic sensory perception in Phycomyces may filter out cytoplasmic light flicker and may not require strong contrasting regions within the lens profile to detect light direction.     

What do we know about the non-uniform perception of a phototropic stimulus in Phycomyces?

For a comprehensive study of phototropism in sporangiophores of the fungus Phycomyces, quantitative treatment of spatial aspects is necessary. The first step in quantifying spatial factors of phototropic signal processing is the elucidation of the non-uniform light profile, predominantly caused by a lens effect in the cylindrical body of the sporangiophore. Herein we compare recently presented theoretical and experimental studies of light profiles. Errors and ambiguities arising from instrumental limitations and arbitrary assumptions are revealed. On the other hand, by combining theoretical and experimental results we have been able to select out the reliable information, which can now be applied in phototropic studies.Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Desensitization by red and blue light of phototropism in maize coleoptiles

The effects of pretreatments with red and blue light (RL, BL) on the fluence-response curve for the phototropism induced by a BL pulse (first positive curvature) were investigated with darkadapted maize (Zea mays L.) coleoptiles. A pulse of RL, giving a fluence sufficient to saturate phytochrome-mediated responses in this material, shifted the bell-shaped phototropic fluence-response curve to higher fluences and increased its peak height. A pulse of high-fluence BL given immediately prior to this RL treatment temporarily suppressed the phototropic fluence-response curve, and shifted the curve to higher fluences than induced by RL alone. The shift by BL progressed rapidly compared to that by RL. The results indicate (1) that first positive curvature is desensitized by both phytochrome and a BL system, (2) that desensitization by BL occurs with respect to both the maximal response and the quantum efficiency, and (3) that the desensitization responses mediated by phytochrome and the BL system can be induced simultaneously but develop following different kinetics. It is suggested that theses desensitization responses contribute to the induction of second positive curvature, a response induced by prolonged irradiation.Abbreviations BL blue light - RL red light CIW-DPB Publication No. 1001     

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.     

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.     

The role of the apex in the phototropic curvature of Avena coleoptiles: positive curvature under conditions of continuous illumination

The differential growth causing second positive phototropic curvature in intact, black-capped and decapitated Avena coleoptiles has been measured. In all cases the curvature is brought about by a cessation in growth of the illuminated side. The fact that shading the apex does not significantly alter the initial steps of differential growth means that the subapical zones can perceive and respond to unilateral illumination. Decapitation significantly reduces coleoptile growth, especially in the most apical zone. However, the fact that differential growth is still evident in the other zones of decapitated coleoptiles within 30 min of unilateral illumination requires one to conclude that the apex cannot be controlling the differential growth in those basal zones.     

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.     

Kinetic modelling of phototropism in maize coleoptiles

Blue-light-induced phototropism of maize (Zea mays L.) coleoptiles was studied with a view to kinetic models. Red-light-grown plants were used to eliminate complication arising from the activation by blue light of phytochrome-mediated phototropism. In the first part, mathematical models were developed to explain the phototropic fluence-response data, which were obtained for the responses induced by a single unilateral pulse (30 s) and those induced by a unilateral pulse (30 s) given immediately after a bilateral pulse (30 s, fixed fluences). These data showed bell-shaped fluence-response curves, characteristic of first positive curvature. Modelling began with the assumptions that the light gradient plays a fundamental role in phototropism and that the magnitude of the response is determined by the gradient, or the concentration difference, in a photoproduct between the irradiated and the shaded sides of the tissue. Minimal mathematical models were then derived, by defining chemical kinetics of the photoreaction and introducing the minimum of parameters needed to correlate the incident fluencerate to the functional fluence-rates within the tissue, the functional fluence-rate to the rate constant of the photoreaction, and the photoproduct concentration difference to the curvature response. The models were tested using a curve-fitting computer program. The model obtained by assigning first-order kinetics to the photoreaction failed to explain the fluence-response data, whereas application of second-order kinetics led to a successful fit of the model to the data. In the second part, temporal aspects of the photosystem were examined. Experimental results showed that a high-fluence bilateral pulse eliminated the bell-shaped fluence-response curve for an immediate unilateral pulse, and that the curve gradually reappeared as the time for unilateral stimulation elapsed after the bilateral pulse. The model based on a second-order photoreaction could be extended to explain the results, with assumed changes in two components: the concentration of the reactant for the photoproduct, and the light-sensitivity of the reaction. The reactant concentration, computed with the curvefitting program, showed a gradual increase from zero to a saturation level. This increase was then modelled in terms of regeneration of the reactant from the photoproduct, with an estimated first-order rate constant of about 0.001·s^-1. The computed value for the constant reflecting the light-sensitivity showed a sharp decline after the high-fluence pulse, followed by a gradual return to the initial level. From these analytical results, the appearance of second positive curvature was predicted.Abbreviations FPC first positive curvature - SPC second positive curvature CIW-DPB publication No. 884     

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1 mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early develpment. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. This increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.     

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