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     

Effects of light and temperature on sporangiophore initiation in Pilobolus crystallinus (Wiggers) Tode

Sporangiophore initiation in Pilobolus crystallinus grown in white light was induced by either a dark or a low-temperature treatment. The period of darkness necessary to induce sporangiophore initiation was shortened by lowering the temperature. Arrhenius plots for the sporangiophore-suppressing reaction in both light and darkness consisted of two straight lines with a Q₁₀ of about 2 at lower temperatures and 8–11 at higher temperatures. The temperature at which the Q₁₀ changed was the lower, the higher the fluence rate: 14° C at 8 W/m², 19.5° C at 0.24 W/m² and 24.5° C in darkness. Possible interpretations of these results are briefly presented.Abbreviations %SP percentage of trophocysts initiating sporangiophores - D50% duration of treatment required to 50% sporangiophore initiation     

Phototropic fluence-response relations for Avena coleoptiles on a clinostat

Phototropism of Avena sativa L. has been characterized using a clinostat to negate the gravitropic response. The kinetics for development of curvature was measured following induction by a single pulse of blue light (BL), five pulses of BL at 20-min intervals, and this same pulsed-light regime following a 2-h red light (RL) pre-irradiation. A final curvature of about 14° is expressed within 180 min following the single pulse; a final curvature of about 62° in about 240 min following five pulses without pre-irradiation; and a curvature of over 125° in 360 min following five pulses after the RL pre-irradiation. For seedlings not pre-irradiated, the final curvature to five pulses of BL at a total fluence of 9.4 pmol·cm^-2 increases with time of darkness between pulses up to 15 min; with seedlings pre-irradiated with RL, curvature increased more slowly with time of darkness between pulses to a maximum at 35 min. The final curvature induced by a constant fluence of 9.4 pmol·cm^-2 increases linearly with time between the first pulse and last pulse of a five-pulse sequence. The curvature induced by a single BL pulse with a 5-min RL co-irradiation increases with fluence to a maximum of about 60° at about 10 pmol·cm^-2, and then decreases to 0° at about 200 pmol·cm^-2. Curvature induced by five BL pulses following a 2-h RL pre-irradiation increased with fluence from a threshold of about 0.05 pmol·cm^-2 to a maximum of 90° at about 10 pmol·cm^-2, and then gradually decreased with fluence to 50° at 1 000 pmol·cm^-2. Based on these data, it is concluded that the initial photoproduct formed by a BL pulse has a limited lifetime, that there is a kinetic limitation downstream of the photoreceptor pigment for phototropism, and that the additivive effect of pulsed BL is distinct from the potentiating effect of RL on phototropism. Thus, any degree of curvature from 0° to over 90° may be induced by a fluence in the ascending arm of what is traditionally called the first positive phototropic response.Abbreviations BL blue light - RL red light     

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.     

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.     

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.     

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     

Phototropism of rice (Oryza sativa L.) coleoptiles: fluence-response relationships, kinetics and photogravitropic equilibrium

Phototropism of rice (Oryza sativa L.) coleoptiles induced by unilateral blue light was characterized using red-light-grown seedlings. Phototropic fluence-response relationships, investigated mainly with submerged coleoptiles, revealed three response types previously identified in oat and maize coleoptiles: two pulse-induced positive phototropisms and a phototropism that depended on stimulation time. The effective ranges of fluences and fluence rates were comparable to those reported for maize. Compared with oats and maize, however, curvature responses in rice were much smaller and coleoptiles straightened faster after establishing the maximal curvature. When stimulated continuously, submerged coleoptiles developed curvature slowly over a period of 6 h, whereas air-grown coleoptiles, which showed smaller phototropic responsiveness, established a photogravitropic equilibrium from about 4 h of stimulation. The plot of the equilibrium angle against log fluence rates yielded a bell-shaped optimum curve that spanned over a relatively wide fluence-rate range; a maximal curvature of 25° occurred at a fluence rate of 1 μmol · m⁻² · s⁻¹. This optimum curve apparently reflects the light sensitivity of the steady-state phototropic response. Received: 28 June 1996 / Accepted: 30 July 1996     

Mutants of Arabidopsis thaliana with altered phototropism

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and second positive phototropism. However, while the amplitude for first positive phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in first positive phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 mol·m^-2 to 2700 mol·m^-2, but shows normal gravitropism. Strain JK345 shows no first positive phototropism, and reduced gravitropism and second positive phototropism. Strain JK229 shows no measurable first positive phototropism, but normal gravitropism and second positive phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. first positive and second positive phototropism contain at least one common element; and 3. first positive phototropism can be substantially altered without any apparent alteration of second positive phototropism.Abbreviation WT wild-type     

Effects of microbeam light on growth and phototropism ofPilobolus crystallinus sporangiophores

A small blue-light beam (50 μm in diam) was used to examine light-growth response and phototropism inPilobolus crystallinus sporangiophores. Continuous irradiation by microbeam of a region 100–150 μm from the apex promoted the growth of a dark-adapted sporangiophore for about 15 min after a lag period of 1–2 min. After the promotion, the growth rate fell below that before the irradiation. Irradiation of the apex of sporangiophore slightly promoted the growth but strongly inhibited the growth after the promotion. A smaller light beam (10 μm in diam) applied continuously at grazing incidence along one side of the sporangiophore caused bending toward the shaded side, implying that the irradiated side grew more rapidly than the shaded side and that the lens effect is involved in the phototropism of young sporangiophores ofP. crystallinus. The involvement of the lens effect was confirmed by the fact that a carotenoid-less mutant was 1.5–2 times more sensitive to unilateral blue light than the wild type, probably because of a smaller intracellular light attenuation during passage through the mutant cell.     

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.     

Redistribution of growth during phototropism and nutation in the pea epicotyl

First positive phototropism of the third internode of intact, 5-d-old pea (Pisum sativum L.) seedlings, grown under continuous, dim red light, showed maximal response following a photon fluence of 3 mol·m^-2 blue light. Greater or lesser fluences (with irradiation time 100 s or less) caused less bending, no response being detectable above 300 or below 0.03 mol·m^-2. Bilateral irradiation with blue light caused no detectable inhibition of growth rate over that range of fluences. The linear nutation of the pea third internode was shown to be driven by a balanced oscillation of growth rate such that the overall growth rate was little changed during the oscillation. Phototropic stimulation changed neither the amplitude nor the period of nutation. Nutation and phototropism probably regulate growth independently. Phototropism in response to the optimal blue light fluence was caused by concomitant depressed growth on the irradiated side and stimulated growth on the shaded side of the bending internode. These results are consistent with the Cholodny-Went hypothesis which states that unilateral blue light induces a lateral redistribution of a growth regulator.Abbreviations R red light - BL blue light Carnegie Institution, Department of Plant Biology paper No. 921     

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.     

Iron and temperature as sporangiospore germination factors of Pilobolus longipes

Sporangiospores of Pilobclus longipes germinated on a medium containing ascorbate and FeSO₄, but neither ascorbate nor FeSO₄ alone caused spores to germinate. The iron chelates (hemin, coprogen, and ferrichrome) that are known to promote mycelial growth of this and other species of Pilobolus had little or no effect on spore germination, suggesting that under these conditions dormant spores are unable to reduce iron III.Regardless of the medium used, maximum germination required treatment at two temperatures. The early stage of germination, spherical growth, was favored by treatment for several hours at about 38°C while optimum germ tube formation required incubation at lower temperatures (25°C). Under most conditions the requirement for a heat treatment was nearly absolute.When the iron-ascorbate and the heat treatments were separated it was found that they need not be applied simultaneously provided that iron and ascorbate are given first. Spores that were heated first and then given iron and ascorbate at lower temperatures did not germinate. Apparently dormancy of these spores is broken by available iron but a heat treatment is usually required to complete the germination process.     

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.     

Photoperception sites for phytochrome-mediated phototropism of maize mesocotyls

The major site of photoperception for phytochrome-mediated phototropism of maize (Zea mays L.) mesocotyls was identified to be within the bending zone of the mesocotyl.Abbreviations FR far-red light - R red light C.I.W.-D.P.B. Publication No. 854     

Bioprospecting for novel oxylipins in fungi: The presence of 3-hydroxy oxylipins in Pilobolus

As previously found in various members of the Mucorales, 3-hydroxy oxylipins in Mucor genevensis are associated with the sporangia, i.e. mainly the columella structure and between aggregating sporangiospores. To determine if this phenomenon is also true in distantly related members, the mucoralean fungus Pilobolus was examined. This fungus is characterized by relatively large sub sporangial-columella structures which actively eject sporangia in a sticky liquid for attachment onto herbage surrounding its growth medium – in this case horse dung. Strikingly, this fungus produced a novel oxylipin i.e. a 3-hydroxy monounsaturated fatty acid, possibly a nonenoic acid, which is mainly associated with the sub sporangial-columella structure and aggregating sporangiospores. The specificity of the antibody against 3-hydroxy oxylipins used in immunofluorescence mapping of the mucoralean fungi, was further confirmed in the yeast, Saccharomycopsis malanga which produces 3-hydroxy palmitate in crystal form. These crystals occur between aggregating yeast cells. On the basis of the available data, we hypothesize that 3-hydroxy oxylipins probably function as adhesives, attaching fungal cells to each other or to other surfaces through entropic based hydrophobic forces and/or hydrogen bonds.     

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     

Phytochrome-mediated phototropism in maize seedling shoots

Unilateral irradiation with red light (R) or blue light (BL) elicits positive curvature of the mesocotyl of maize (Zea mays L.) seedlings raised under R for 2 d from sowing and kept in the dark for 1 d prior to curvature induction. The fluenceresponse curve for R-induced mesocotyl curvature, obtained by measuring curvature 100 min after phototropic induction, shows peaks in two fluence ranges, designated first positive range (from the threshold to the trough), and second positive range (above the trough). The fluence-response curve for BL is similar to that for R but shifted two orders of magnitude to higher fluences. Blue light elicits the classical first positive curvature of the coleoptile, whereas this response is not found with R. Positive mesocotyl curvature induced by either R or BL is eliminated by R given from above just before the unilateral irradiation, whereas BL-induced coleoptile curvature is not eliminated. The above results collectively offer evidence that phototropic curvature of the mesocotyl is induced by R-sensitive photosystem(s). Mesocotyl curvature in the second positive range is reduced by vertical far-red light (FR) applied after phototropic induction with R, but is not affected by FR applied before R. Unilateral irradiation with FR following vertical irradiation with a high R fluence leads to negative curvature of the mesocotyl. It is concluded that mesocotyl curvature in the second positive range results from a gradient in the amount of the FR-absorbing form of phytochrome (Pfr) established across the plant axis. Mesocotyl curvature in the first positive range is inhibited by vertical FR given either before or after phototropic induction with R. Since the FR used here is likely to produce more Pfr than the very low fluences of R eliciting the mesocotyl curvature in the first positive range, it is assumed that FR reduces the response in this case by adding Pfr at both sides of the plant axis. By rotating seedlings on a clinostat with its axis horizontal, the kinetics of mesocotyl curvature can be studied in the absence of a counteracting gravitropic response. On the clinostat, the R-induced mesocotyl curvature develops after a lag, through two successive phases having different curvature rates, the late phase is slower than the early phase. Negative curvature of the coleoptile can be induced by either R or BL; the BL-induced negative curvature is found at fluences higher than those giving positive curvature. The clinostat experiments show that the negative coleoptile curvature induced by either R or BL is a gravitropic compensation for positive mesocotyl curvature.Abbreviations BL blue light - FR far-red light - Pfr phytochrome in the far-red-absorbing form - Pr phytochrome in the red-absorbing form - R red light C.I.W.-D.P.B. Publication No. 824     

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)