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.     

Second positive phototropism in the Avena coleoptile

A method has been developed whereby the second positive phototropism can be observed separately from the first positive and negative phototropic responses which also occur in oat coleoptiles. Although the second positive phototropic response has often been referred to as the base response, photoreception for it is shown to occur mainly in the apical 3 mm of the coleoptile. The Bunsen-Roscoe reciprocity law, so typical of first positive phototropism, does not apply to the second positive responses, and the amount of curvature increases linearly with the duration of the stimulus. However, although this linear proportionality between stimulus duration and response is the major factor determining response at all intensities tested, the intensity of the stimulus does influence the response somewhat. The action spectrum for the response shows no activity above 510 nm and has peaks at 375 and 450 nm. In all but one particular it closely resembles the action spectrum for the first positive phototropism, and it is concluded that the same, or similar, pigments may well be the photoreceptors for both types of response. The identity of this blue light absorbing pigment is not known.     

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)     

Negative phototropic response of rhizoid cells in the fern Adiantum capillus-veneris

In general, phototropic responses in land plants are induced by blue light and mediated by blue light receptor phototropins. In many cryptogam plants including the fern Adiantum capillus-veneris, however, red as well as blue light effectively induces a positive phototropic response in protonemal cells. In A. capillus-veneris, the red light effect on the tropistic response is mediated by phytochrome 3 (phy3), a chimeric photoreceptor of phytochrome and full-length phototropin. Here, we report red and blue light-induced negative phototropism in A. capillus-veneris rhizoid cells. Mutants deficient for phy3 lacked red light-induced negative phototropism, indicating that under red light, phy3 mediates negative phototropism in rhizoid cells, contrasting with its role in regulating positive phototropism in protonemal cells. Mutants for phy3 were also partially deficient in rhizoid blue light-induced negative phototropism, suggesting that phy3, in conjunction with phototropins, redundantly mediates the blue light response.     

Influence of phototropic response of spore germ tubes on infection process inColletotrichum lagenarium andBipolaris oryzae

The germ tubes ofColletotrichum lagenarium showed negative phototropism to UV-blue (300–520 nm) and far-red (>700 nm) regions with maximum in the near ultraviolet (NUV) region, while monochromatic radiations of 575–700 nm (yellow-red region) induced positive phototropism with maximum in the red region. Green light (520–575 nm) was ineffective. Negative phototropism-inducing wavelength regions inhibited germ tube growth and positive phototropism-inducing wavelength regions promoted it significantly.Bipolaris oryzae did not show any phototropic response and light did not affect the germ tube growth. These results indicate that the lens effect, in combination with the light growth reaction and light growth inhibition, is the mechanism of the phototropism of germ tubes ofC. lagenarium. NUV radiation, which induced negative phototropism ofC. lagenarium, promoted appressorium formation, while red light, which induced positive phototropism, suppressed it significantly. In the case ofB. oryzae, light did not affect the infection structure formation. These results indicate that negative phototropism of germ tubes ofC. lagenarium favors the infection process by facilitating the contact of the tips of germ tubes with the host surface, while positive phototropism has the opposite effect.     

Action Spectrum in the Ultraviolet Region for Phototropism of Bryopsis Rhizoids

The phototropic response of the rhizoid of the marine coenocyticgreen alga Bryopsis plumosa to ultraviolet light (250–350nm) was investigated. The rhizoid exhibited negative bendingthat was due to bulging upon absorption of light in the UV region,as well as in the visible region, of the spectrum. The negativebending might not be a result of the inhibition of growth onthe irradiated side of the apical hemisphere by UV irradiationbecause growth inhibition was observed after bending had reacheda maximum within one to two hours. The action spectrum obtainedfrom fluence rate-response curves had a pronounced peak at 260nm and a small peak at 310 nm. The quantum effectiveness at260 nm was about five times that in the visible region. Phenylaceticacid (PAA), a potent inhibitor of flavin photoreactions, inhibitedthe phototropic response to both UV light and blue light withoutany obvious effect on tip growth. The inhibition of the phototropicresponse to blue light by PAA was partially overcome by rinsingthe alga with riboflavin-containing medium, which result suggeststhe involvement of flavins in the phototropism of Bryopsis rhizoids. (Received February 6, 1995; Accepted June 19, 1995)     

Role of carotenoids in the phototropic response of corn seedlings

The herbicide, 4 chloro-5-(methylamino)-2-(α,α,α,-trifluoro-m-tolyl)-3 (2H)-pyridazinone (SAN 9789), which blocks the synthesis in higher plants of colored carotenoids but not of flavins, was used to examine the involvement of carotenoids in corn seedling phototropism. It was concluded that “bulk” carotenoids are not the photoreceptor pigment based on the results that increasing concentrations of SAN 9789 (up to 100 micromolar) did not alter the phototropic sensitivity to 380 nanometers light (using geotropism as a control) and did not increase the threshold intensities of fluence response curves for both 380 and 450 nanometers light even though carotenoid content was reduced to 1 to 2% of normal. SAN 9789 treatment, however, did reduce seedling sensitivity toward 450 nanometers light indicating that carotenoids are involved in phototropism. Carotenoids, which are located mainly in the primary leaves, may act in phototropism as an internal screen, enhancing the light intensity gradient across the seedling and thus contributing to the ability of the seedling to perceive light direction. These results indicate that the action spectra for phototropic responses can be significantly affected by the absorbance of screening pigments in vivo thus altering its shape from the in vitro absorption spectrum of the photoreceptor pigment.     

Control of hypocotyl gravitropism by photochrome in a dicotyledonous seedling (Sesamum indicum L.)

Abstract The present study was prompted by the question as to whether the strong effect of red and far-red light treatments on blue-light-mediated phototropism in the sesame (Sesamum indicum L.) hypocotyl (Woitzik & Mohr, 1988) should be attributed in part to changes initialed by light in the gravitropic counter-response. Light treatments, operating through phytochrome, do indeed strongly affect the gravitropic response. However, the direction of the light effect is the same in gravitropism, as in phototropism. Thus, the gravitropic counter-response leads to an underestimate, rather than an overestimate, of the importance of phytochrome action on phototropic responsiveness. The effect of red and far-red light, operating via phytochrome, on the gravitropic response of the sesame hypocotyl could be studied in the present paper without any interference due to phototropism or light control of longitudinal growth. It was found that the effects of red and far-red pretreatments (given prior to the onset of the stimulus) as well as the action of simultaneously applied red or far-red light (simultaneous to the phototropic or gravitropic stimulus) are very similar in both phototropism and gravitropism. In particular, the seedling is capable of superimposing information about the actual light conditions during bending on the ‘memory’ it has about the light conditions prior to the onset of phototropism or gravitropic stimulation, This striking similarity between the phototropic and gravitropic responses possibly indicates that phytochrome affects the signal-response-chain at a relatively late stage, after the phototropic and the gravitropic signal-response chains have merged. From a teleonomic point of view the action of red and far-red light on phototropic, as well as gravitropic, responsiveness can be conceived as part of a shade escape strategy.     

Action spectrum of negative phototropism in Boergesenia forbesii

The rhizoid of Boergesenia forbesii, a gigantic unicellulargreen alga, exhibited negative phototropism. The bending processat 32?C followed Weber-Fechner's law and Bunsen- Roscoe's law.The minimum light energy inducing the phototropic bending was30 J.m^–2 at 467 nm and 32?C. The action spectrum of thisnegative phototropism had two distinct peaks at 380 and 443nm, with shoulders at 430 and 470 nm and a trough around 410run. Light of wavelengths longer than 502 nm was ineffective.The structure of the spectrum agrees well with that reportedfor the positive phototropism of Avena coleoptiles and otherplant parts. (Received February 24, 1979; )     

The role of cotyledons in phototropism of de-etiolated seedlings

Simulated phototropic curvatures caused by differential masking of the cotyledons of de-etiolated seedlings exposed to white light are unconnected with true phototropism. In Cucumis sativus L. and Helianthus annuus L. such curvatures result from a red-light-induced inhibition coming from the exposed cotyledon. True phototropic bending in these species under long-term exposure to fairly high irradiances (as in nature) is a response to blue light. It occurs even when cotyledons are completely covered. These results show that the cotyledons do not perceive the phototropic stimulus and need not be illuminated for phototropism to occur.     

Phytochrome modulation of blue-light-induced phototropism

Red light enhances hypocotyl phototropism toward unilateral blue light through a phytochrome‐mediated response. This study demonstrates how the phytochromes modulate blue‐light‐induced phototropism in the absence of a red light pre‐treatment. It was found that phytochromes A, B, and D have conditionally overlapping functions in the promotion of blue‐light‐induced phototropism. Under very low blue light intensities (0.01 µmol m^?2 s^?1) phyA activity is necessary for the progression of a normal phototropic response, whereas above 1.0 µmol m^?1 s^?2 phyB and phyD have functional redundancy with phyA to promote phototropism. PhyA also contributes to attenuation of phototropism under high fluence rates of unilateral blue light, which was previously shown to be dependent on the phototropins and cryptochromes. From these results, it appears that phytochromes are required to develop a robust phototropic response under low fluence rates, whereas under high irradiances where phototropism may be less important, phyA suppresses phototropism.     

Blue light promotes ionic current influx at the growing apex ofVaucheria terrestris

Irradiation of the growing apex of the algaVaucheria terrestris Götz var.terrestris with blue light (BL), which causes a transient acceleration of growth, also causes a large transient increase in inwardly directed current, which was monitored with a vibrating probe. The growing apex is normally the site of an inward current, and the surface of the non-growing, basal part of the coenocytic cell the site of an outward current. Irradiation of the apex causes only a slight increase in current efflux at the basal part of the cell. The BL-promoted current influx at the apex (BLCI) usually starts within 10 s after the onset of irradiation, preceding the light-growth response. With BL pulses shorter than 3 min, the BLCI reaches a maximum in about 3 min, and then declines to its original value over the next 3 min. If the BL pulse is longer than 3 min, the BLCI continues until the light is turned off. The threshold energy of the BLCI with broad-band BL is 2–5 J·m^-2, i.e. smaller than for both the light-growth response and phototropic response. The maximum BLCI reaches a value of approx. 5 A·cm^-2, equivalent to an influx of 50 pmol·cm^-2·s^-1 of monovalent cations. The effect of red light (RL) is completely different from that of BL: it either causes increases in the inward current of less than 0.3 A·cm^-2, or a transient decrease of current. Furthermore, the direction of the RL-induced change is always the same at the apex and trunk, indicating the participation of photosynthesis. Our results indicate that the BLCI is kinetically and spatially related to the light-growth response and the phototropic bending ofVaucheria. It seems to be a necessary step for the phototropic bending.Abbreviations APW artificial pond water - BL blue light - BLCI blue-light-induced current influx - LGR light-growth response - RL red light     

System analysis of Phycomyces light-growth response. Photoreceptor and hypertropic mutants.

The light-growth responses of Phycomyces behavioral mutants, defective in genes madB, madC, and madH, were studied with the sum-of-sinusoids method of system identification. Modified phototropic action spectra of these mutants have indicated that they have altered photoreceptors (P. Galland and E.D. Lipson, 1985, Photochem. Photobiol. 41:331). In the two preceding papers, a kinetic model of the light-growth response system was developed and applied to wild-type frequency kernels at several wavelengths and temperatures. The present mutant studies were conducted at wavelength 477 nm. The log-mean intensity was 6 X 10(-2)W m-2 for the madB and madC night-blind mutants, and 10(-4)W m-2 for the madH hypertropic mutant. The prolonged light-growth responses of the madB and madC mutants are reflected in the reduced dynamic order of their frequency kernels. The linear response of the hypertropic mutant is essentially normal, but its nonlinear behavior shows modified dynamics. The behavior of these mutants can be accounted for by suitable modifications of the parametric model of the system. These modifications together support the hypothesis that an integrated complex mediates sensory transduction in the light responses and other responses of the sporangiophore.     

Evidence for a phytochrome-mediated phototropism in etiolated pea seedlings

Entirely etiolated pea seedlings (Pisum sativum, L. cv Alaska) were tested for a phototropic response to short pulses of unilateral blue light. They responded with small curvatures resembling in fluence-dependence and kinetics of development a phytochrome-mediated phototropic response previously described in maize mesocotyls. Irradiations from above with saturating red or far-red light, either immediately before or after the unilateral phototropic stimulus, strongly reduced or eliminated subsequent positive phototropic curvature. Only blue light from above, however, entirely eliminated curvature at all fluences of stimulus. It is concluded that the phototropism is primarily a result of phytochrome action.     

Root phototropism: from dogma to the mechanism of blue light perception

In roots, the “hidden half” of all land plants, gravity is an important signal that determines the direction of growth in the soil. Hence, positive gravitropism has been studied in detail. However, since the 19th century, the response of roots toward unilateral light has also been analyzed. Based on studies on white mustard (Sinapis alba) seedlings, botanists have concluded that all roots are negatively phototropic. This “Sinapis-dogma” was refuted in a seminal study on root phototropism published a century ago, where it was shown that less then half of the 166 plant species investigated behave like S. alba, whereas 53% displayed no phototropic response at all. Here we summarize the history of research on root phototropism, discuss this phenomenon with reference to unpublished data on garden cress (Lepidium sativum) seedlings, and describe the effects of blue light on the negative bending response in Thale cress (Arabidopsis thaliana). The ecological significance of root phototropism is discussed and the relationships between gravi- and phototropism are outlined, with respect to the starch-statolith-theory of gravity perception. Finally, we present an integrative model of gravi- and blue light perception in the root tip of Arabidopsis seedlings. This hypothesis is based on our current view of the starch-statolith-concept and light sensing via the cytoplasmic red/blue light photoreceptor phytochrome A and the plasma membrane-associated blue light receptor phototropin-1. Open questions and possible research agendas for the future are summarized.     

Action spectra for photogrowth and phototropism in protonemata of the moss Physcomitrium turbinatum

Summary Protonemata of Physcomitrium were grown in a sucrose-mineral nutrient, liquid medium. Even in this medium containing organic nutrient, the growth rate of lateral branch, chloronemal filaments showed a light dependence which was linear with log intensity. Intensities necessary to give a constant growth rate (45 /1.75 hrs.) were determined at selected wavelengths. The resulting action spectrum paralleled the in-vivo absorption spectrum of a single filament in the red region, showing a major peak at 680 nm. Growth rate and absorption approached zero in the far-red (730 nm). However, significant growth activity occurred at 365–400 nm where absorption was low, and negligible growth was found at 440–500 nm where absorption was high.The action spectrum for the positive, directional photo-orientation of growth was determined by the null-point method in which the effectiveness of each selected wavelength was compared to a 665-nm standard in simultaneous, bilateral irradiation. In contrast to growth, the major peak of phototropic activity was found at 730 nm with significant activity extending to 800 nm. A minor peak was at 680 nm. There was some activity in near ultraviolet but not at longer blue wavelengths.It is concluded that the blue-absorbing system responsible for phototropism in virtually all other groups of plants is inactive or absent in Physcomitrium. Instead growth and orientation seem to be dependent upon an interaction between the photosynthetic and phytochrome systems. Further, the data suggest that the physiological activity of phytochrome in photo-orientation of growth does not derive from a certain amount of P_fr or P_fr/P_r ratio but rather it derives from the simultaneous excitation and consequent cycling of P_r and P_fr.Published with the approval of the Secretary of the Smithsonian Institution.This work was carried out under partial support by the U.S. Atomic Energy Commission under Contract AT(30-1)2373.     

Desensitization and recovery of phototropic responsiveness in Arabidopsis thaliana.

Phototropism is induced by blue light, which also induces desensitization, a partial or total loss of phototropic responsiveness. The fluence and fluence-rate dependence of desensitization and recovery from desensitization have been measured for etiolated and red light (669-nm) preirradiated Arabidopsis thaliana seedlings. The extent of desensitization increased as the fluence of the desensitizing 450-nm light was increased from 0.3 to 60 micromoles m-2 s-1. At equal fluences, blue light caused more desensitization when given at a fluence rate of 1.0 micromole m-2 s-1 than at 0.3 micromole m-2 s-1. In addition, seedlings irradiated with blue light at the higher fluence rate required a longer recovery time than seedlings irradiated at the lower fluence rate. A red light preirradiation, probably mediated via phytochrome, decreased the time required for recovery from desensitization. The minimum time for detectable recovery was about 65 s, and the maximum time observed was about 10 min. It is proposed that the descending arm of the fluence-response relationship for first positive phototropism is a consequence of desensitization, and that the time threshold for second positive phototropism establishes a period during which recovery from desensitization occurs.     

OCTAHEDRAL CRYSTALS IN PHYCOMYCES. II

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the β-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265–285 nm, 350–380 nm, and 450–470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".