MEASUREMENT OF PHYCOMYCES LIGHT RESPONSES

EDWARD D. LIPSON (edlipson@syr.edu)
Department of Physics, Syracuse University, Syracuse, New York 13244-1130, USA

PAUL GALLAND (galland@nws.biologie.uni-marburg.de)
Fachbereich Biologie, Universität Marburg, D-35032 Marburg, Germany

In Phycomyces. E. Cerdá-Olmedo and E. D. Lipson (eds.), 1987, p. 367-373. CSH Laboratory Press, Cold Spring Harbor, NY, USA.

In this appendix, we outline the methods for measuring the blue-light responses that occur in the sporangiophores (phototropism and light-growth response) and in the mycelium (photocarotenogenesis, photophorogenesis, and photosporangiogenesis). Appendix 8 deals with pertinent light sources, calibrations, and radiometric quantities.

Sporangiophore Responses

Phototropism

In the most common type of phototropism measurement, the angle of the sporangiophores is recorded as a function of time in response to a unilateral light stimulus. An important consideration in such measurements is the preadaptation condition of the sporangiophore. One way to adapt the sporangiophore to symmetrical light is to rotate it on a turntable at about six revolutions per minute while it is exposed to a single horizontal light beam. The unilateral stimulus begins when the rotation is stopped. A phototropic response will then ensue after a latent period of several minutes. Alternatively, one can adapt the sporangiophore to bilateral symmetric illumination and then (at time zero) extinguish one of the beams and set the other beam to a new intensity (e.g., double the initial intensity of each of the bilateral beams or, alternatively, lower the intensity for dark adaptation studies by the phototropic delay method; see below). Instead of rotating the sporangiophore, a mirror can be placed on the distal side of the sporangiophore (with respect to the light beam) so that the collimated beam is reflected back (Galland and Russo 1984). Then, at time zero, the mirror is removed and the intensity of the light is adjusted.
To measure the bending angle of the sporangiophore, the simplest approach is to use a horizontal measuring microscope (Gaertner Scientific, Chicago, Illinois) with a goniometer attachment. For observation, the sporangiophore is back-illuminated with a red safelight (e.g., a tungsten lamp with red acrylic plastic, type S10218, Mitsubishi Rayon Company, available in large sheets from Payne Glass Company, Pasadena, California). Except for this safelight and the illuminator for the adaptation and stimulus, the specimen is otherwise observed within the dark room.
An alternative to using a dark room is to enclose the sporangiophore in a chamber that has front and rear windows (made of this special red plastic, or other suitable cutoff filter made of plastic or glass) and side ports for stimulus illumination. Such a setup may be located conveniently in a normally illuminated laboratory environment, provided care is taken to exclude stray light from the ports.
With the horizontal microscope, the angle is recorded at time intervals of 30 seconds to 2 minutes using a stopwatch or electronic timer. In this case, the experimenter writes down the angle and later graphs the results manually. Such a graph is usually characterized by a latent period during which there is no significant bending, followed by a period where bending gradually increases and reaches a steady bending rate of about 3°/min for approximately 20 or 30 minutes. By extrapolation of this linear bending region back to the original baseline (also extrapolated), the phototropic delay, or latency, can be measured as the interval between the stimulus and the intersection of these two asymptotes.
A more convenient way to measure phototropism, without the observer being continuously present, is to employ a time-lapse video cassette recorder (Galland et al. 1984). The sporangiophore is observed by a video camera and the images are recorded on videotape with a time-lapse ratio of about 30:1. On playback, the angle is recorded as a function of time. (Note: The tape counter on video recorders is not useful for this purpose because the count does not increase in direct proportion to the time, but rather to the rotation of a takeup reel). One way to show the time on each frame is to employ a video time-display generator; however, these devices are expensive and the procedure of starting and stopping the video tape is still time consuming and will reduce the lifetime of the machine.
A simple, efficient alternative is to employ an "electronic protractor" consisting simply of a precision rotary potentiometer (Lipson and Hader 1984). If a fixed voltage (e.g., 5 volts) is applied across the terminals of the potentiometer, then the voltage on the tap of the potentiometer is directly proportional to the angle of rotation. By connecting a transparent ruler to the shaft of the potentiometer, one can view the sporangiophore image on a video monitor, rotate the ruler continuously to remain parallel to the upper part of the sporangiophore, and thereby produce a precise, smooth record on the chart recorder of the bending angle as a function of time. Moreover, by this method, several successive runs can be played back without interruption .

Photogravitropism

The equilibrium between phototropism and gravitropism provides a convenient assay for the phototropic sensitivity as a function of absolute light intensity. Two similar approaches have been used to quantify this light dependence of phototropism (Bergman et al. 1973; Lipson and Terasaka 1981). With a "threshold box," beam splitters are used to provide ten compartments with progressively decreasing light intensities. If the beam splitters are approximately 50% transmitting and 50% reflecting, then this system covers a range of approximately 1000:1. This range may be increased either by introducing neutral density filters between the compartments or by using beam splitters with lower transmission and higher reflection. In such a threshold box, the sporangiophores will bend toward the light beams obtained after reflection from the successive beam splitters. After 6-9 hours of exposure, the angles of the sporangiophores are recorded. These angles may be measured with a simple protractor with or without a magnified rear projection system.
There are two conventional ways to measure the photogravitropism angles. One way is simply to measure the angle of the sporangiophores projected into the plane of the light beam and the vertical. In this case, the holder containing about six vials of sporangiophores is removed, the sporangiophores are observed from the side, and the bending angle from the vertical toward the light is measured (with a protractor). The preferred way, however, is to measure both the polar and the azimuthal angles (aiming errors), which can become considerable (Galland 1983); information is lost if only the projected bending angles are recorded.
Instead of a threshold box, which limits the number of specimens that can be observed in one experiment, an alternative is a long dark room with a point light source (viz. small lamp filament) at one end of the room (Bergman et al. 1973; Lipson et al. 1983). Stations are located that differ successively in intensity by a factor of two. Here, the intensity is reduced not by beam splitters but rather by the inverse square law. The analysis is similar to that in the threshold box, as described above. It is prudent to have suitable enclosures at the stations to suppress winds (anemotropism will occur in response to even slight convection currents); inverted aquarium tanks provide inexpensive transparent enclosures.

Phototropic Balance Measurements

The phototropic balance method is used primarily for action spectroscopy studies (Curry and Gruen 1959; Delbruck and Shropshire 1960; Galland and Lipson 1985). In this case, a standard light source of fixed intensity on one side is used with either broadband blue light or preferably monochromatic blue light. On the other side, the light source is monochromatic with variable wavelength and intensity. Sporangiophores are located at various positions between the two sources, preferably inside an enclosed box to suppress winds. Then, the intensity of the test light is determined which balances the standard intensity, so that sporangiophores do not bend. Preliminary experiments should be conducted to determine approximately the balance condition. The intensity of the test light should then be adjusted so that the balance point is near the midpoint; otherwise, certain systematic errors can arise because the relative efficiency is generally intensity-dependent (Galland and Lipson 1985). A new balance experiment is then performed with a large number of sporangiophores, and the balance point is determined precisely. In such experiments, one should have a precise calibration of the intensity of each light source as a function of position along the box. In phototropic balance experiments, the observer may either record the polar and azimuthal angles of all sporangiophores studied or more simply record only the position of the balance point in the box.

Frozen Sinusoidal and Helical Wave Patterns

For certain photototropism studies, it is convenient to induce a pattern of growth more complex than a simple phototropic bend toward a unilateral light source, so that one can determine whether or not a sporangiophore is able to see a particular intensity. The method described here may also be used as a basis for certain mutant hunts.
A suitable technique was developed (Lipson et al. 1983) both for mutant hunts and for qualitative tests of phototropism with mutant and wild-type strains at different intensities. In this method, two light sources on opposite sides are switched on and off alternately in succession. The period of alternation may be, for example, 2 or 4 hours. In these experiments, the sporangiophore bends first toward one light source and then toward the other. This back-and-forth bending induces a sinusoidal pattern that becomes frozen into the growth of the sporangiophores. This approach is especially advantageous in mass cultures on petri dishes in which transient shadowing effects may give a false indication of phototropism at high intensity. This approach is also used conveniently for tests of phototropism, for example, to determine if heterokaryons or progeny from crosses are sensitive to phototropism at low intensity where certain mad mutants are insensitive (López-Díaz and Lipson 1983). To obtain the alternating illumination, a recycling timer is used to switch the lights. For mass culture analysis, two opposed banks of blue fluorescent lamps are optimal (Lipson et al. 1983).

Light-growth Response

The light-growth response is recorded most simply by means of a horizontal microscope with red-safelight back illumination and symmetrical stimulating lights on either side for observation. To avoid phototropic hunting, it is advisable to use inclined light beams (Delbrück and Reichardt 1956). A filar eyepiece micrometer is used to record the longitudinal position of the top of the sporangium as a function of time, usually at 30-second intervals. After such an experiment, the sporangiophore height may be plotted as a function of time or, by taking successive differences, the growth rate may be plotted as a function of time. Again, this approach may be automated with the help of video equipment.
The best method for measuring light-growth responses or spontaneous growth-rate fluctuations of individual sporangiophores is to use a tracking machine (Foster 1972; Foster and Lipson 1973; Lipson 1975; Lafay and Matricon 1985).

Mycelial Photoresponses

Photocarotenogenesis

Blue light partialiy controls the amount of beta-carotene in mycelium (and sporangiophores as well; however, essentially all experiments are carried out on mycelial preparations). The effect of light depends on the given physiological conditions and can either stimulate anabolism (Bergman et al. 1973; López-Díaz and Cerdá-Olmedo 1980) or inhibit catabolism of beta-carotene (Raugei et al. 1982). Most investigators used mycelial growing on agar plates; studies with liquid cultures are relatively rare (Sandmann and Hilgenberg 1978). Mycelium can be irradiated either continuously or with pulses of relative short duration of a few minutes. The amount of photoinduced beta-carotene can be determined at various times from 20 minutes to 12 hours after irradiation. A typical and often used protocol for beta-carotene determination was described by De la Guardia et al. (1971). Mycelia are peeled off the agar plates and divided into two portions: one for dry weight determination and another for the measurement of beta-carotene. The beta-carotene is extracted by blending a piece of mycelium (about 200 mg dry weight) in a mixer with 20 ml of methanol and 20 ml of petroleum ether. The process is repeated twice, and the combined petroleum ether fractions are centrifuged to remove cell debris. The supernatant is concentrated under a stream of nitrogen and is then chromatographed on a MgO-diatomaceous earth column before the spectra of the colored and uncolored carotenoids are taken. Modifications of the extraction procedure can be found in Ootaki et al. (1973), Sandmann and Hilgenberg (1978), and Raugei et al. (1982). For many purposes, it is unnecessary to separate chromatographically the various carotenoids; it is sufficient to measure the absorption at 448 nm, since no other blue-light-absorbing pigments are extracted by this method.
Light-induced beta-carotene synthesis has been measured also with an in vivo spectrophotometric assay (Jayaram et al. 1979). This assay, however, can be used only for studies on mycelium. The beta-carotene-specific absorption of the mycelium is determined in the following way: Light beams of 455 nm and 633 nm (or nearby wavelengths in the blue and red part of the spectrum) are passed through plates containing a layer of mycelium, and the light transmission is measured with a photodiode below the plate. The photocurrent at 455 nm measures the transmission loss due to beta-carotene accumulation as well as nonspecific absorption and light scattering. It was found that the absorption due to light-induced beta-carotene synthesis (ALlCS) is well described by the following formula: A_LICS = a log I_r + b - log I_b, where a and b are constants, which must be determined anew in each set of experiments and which can differ from strain to strain; I_r and I_b are the photocurrents at 633 nm and 455 nm, respectively. In control experiments, this in vivo technique gave results similar to those obtained by the extraction procedure described above (Jayaram et al. 1979). The experimental setup for this assay is easy to build, as it requires only a small dark box in which the plate with mycelium can be held below the light source and above the photodiode. The box should also contain a slider for the two interference filters, so that the wavelength can be easily switched.

Photophorogenesis

Light induction of sporangiophores has been studied under a variety of conditions. A quantitative analysis is feasible only for a few of them. One method to quantify photophorogenesis is based on the "closed house" effect (Russo 1977). If a number of inoculated shell vials are kept in a closed glass beaker, the number of sporangiophores depends greatly on the ratio of the beaker volume to the surface area of mycelium: for smaller volume per unit area of mycelium, fewer sporangiophores are formed. Short pulses of blue light, given to the mycelium 50 hours after inoculation, can reverse this inhibition, and the number of sporangiophores per vial can be brought back to the normal level. Rather precise fluence-response curves can be made with this method (Galland and Russo 1979).
The production of microphores can be controlled by a variety of environmental stimuli; light inhibits microphorogenesis (Gutierrez-Corona and Cerda-Olmedo 1985). The most efficient method to date for quantifying photoinhibition of microphores and photostimulation of macrophores was developed recently (L.M. Corrochano, pers. comm.). Petri dishes with minimal medium (see Appendix 1) are inoculated with 10⁵ heat-shocked spores (in 2 ml of minimal soft-agar). The petri dishes are kept for 48 hours in darkness and are then irradiated for 2-10 minutes with blue light and returned for an additional 48 hours to darkness before microphores are counted. Macrophores are removed from the plate and their dry weight is determined.

Photosporangiogenesis

To study the effect of light on the formation of sporangia, one must work with a synchronized sporangiophore population. Because sporangiophores do not appear synchronously under normal conditions, the formation of sporangia would also be asynchronous and therefore difficult to study. Stage I sporangiophores can be synchronized by a method described by Russo (1977) and then transferred to glass beakers that are kept tightly sealed. Under these conditions, sporangiogenesis is completed in approximately 40 hours, but it is greatly accelerated by continuous irradiation with broadband blue light. Precise fluence-response curves can be made by this method (Russo et al. 1980).

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