Planta

Planta (1988) 174:309-314

9 Springer-Verlag 1988

Photomorphogenesis in Phycomyces: Dependence on environmental conditions Luis M. Corrochano and Enrique Cerdfi-Olmedo* Departamento de Gen6tica, Facultad de Biologia, Universidad de Sevilla, Sevilla, Spain

Abstract. The production of two kinds of vegetative reproductive structures, microphores and macrophores, by Phycomyces blakesleeanus Bgff. depends on plating density, ventilation, asparagine supply, and illumination. Quantitative determinations of these variables lead us to propose a new experimental system for developmental photobiology: standard plastic Petri plates containing 25 ml minimal medium are inoculated with 105 viable, heat-activated spores and incubated, unpiled and unsealed, at 22 ~ C. After 4 d microphores are counted and macrophores are weighed. Both microphorogenesis and macrophorogenesis are governed by light. Photosensitivity is a developmental phenomenon which occurs 32 to 68 h after inoculation, just before the beginning of vegetative reproduction in the dark controls. The maximum photosensitivity occurs 48 h after inoculation.

Key words: Blue light - Phorogenesis - Photomorphogenesis - Phycomyces - Sporangiophore development.

Introduction

The sporangiophores of the fungus Phycomyces blakesleeanus are aerial hyphae that develop an apical sporangium filled with vegetative spores. Sporangiophores occur in very different sizes. The macrophores grow ten or more centimeters into the air under the guidance of light and other stimuli. The microphores are less than 3 mm long. Many cultures contain both kinds of sporangiophores. Sporangiophore development (phorogenesis) is a complex phenomenon influenced by light, nu* To whom correspondence should be addressed

trients (particularly asparagine), and other environmental variables (Rudolph 1958; Thornton 1972, 1973; Sandmann and Hilgenberg 1979). Light has very marked effects on macrophorogenesis under two different sets of conditions described by Bergman (1972) and Russo (1977). Both experimental systems have practical drawbacks that have hindered their use. Crowding, darkness, low temperatures, and lack of ventilation favor microphorogenesis (Thornton 1972; Guti6rrez-Corona and Cerdfi-O1medo 1985; Ortiz-Castellanos and Guti+rrez-Corona, 1988). Under harsh conditions of cold and poor media, microphorogenesis is inhibited by light (Thornton 1973, 1975); this inhibition is mediated by some of the gene products responsible for the phototropism of the macrophores (L6pezDiaz and Cerdfi-Olmedo 1981). Those conditions are inappropriate for photobiological research because they lead to slow growth and erratic results. We have examined the effect of various environmental conditions on phorogenesis and the time course of the development of photosensitivity. As a consequence, we have defined suitable ,conditions for the study of light effects on Phycomyces development. Material and methods The standard wild-type strain NRRL1555 of Phycomyces blakesleeanus Bgff. was cultured and manipulated as described by Cerdfi-Olmedo (1987). Spores were activated by heat shock (48 ~ C, 15 min) and suspended in melted minimal agar (7 g. 1-1 agar); 2-ml samples were layered on solidified minimal agar (about 25 ml, 15 g.1-1 agar) in standard plastic Petri dishes (internal diameter 8 cm). Because aeration influences phorogenesis, normal ('unsealed') plates were left unpiled and care was taken not to open them. For 'sealed' cultures, two plates were incubated inside a paraffin-sealed plastic box (2.4 1). ' O p e n ' plates were incubated with their lids off in a light-tight wooden box (14 1) with 20 ml water to prevent desiccation. For incuba-

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L.M. Corrochano and E. CerdS~-Olmedo: Morphogenesis in

tion in the light, the cultures were exposed in an air-conditioned room (22 ~ C) to 0.6 W - m -2 white light under a diffusing glass lit by a battery of four fluorescent tubes (F40T1210; Sylvania, Danvers, Mass., USA). For incubation in the dark, the Petri dishes were placed in a cardboard box (87 1) in the same room; results in the cardboard box were identical to those observed in a dark room. Fluence rates were measured (Galland and Lipson 1987) through a KG-1 heat filter (Schott, Mainz, FRG) with a PIN10DP photodiode (United Detector Technology, Hawthorne, Cal., USA) connected to a model 480 picoammeter (Keithley Instruments, Cleveland, Ohio, USA). The photodiode was calibrated with a model E4 thermopile (Eppley Lab., Newport, RI, USA). Microphores carrying visible sporangia were counted under a 40 x stereomicroscope. The total number of microphores per plate was estimated from random samples (at least 200 microphores) counted through a calibrated grid. Macrophores and mycelia were plucked from the plates, dried at 105~ for 14-16 h, and weighed. Macrophores are very hard to count, but, in the conditions of our work, their dry weight is about 120 gg, irrespective of length (Gruen 1959). In the figures, each symbol gives the average results of two to four plates from the same experiment, and different symbols express results from independent experiments.

Results

Phorogenesis in the dark. The production of sporangiophores (microphore numbers and macrophore weight per plate) is largely independent of inoculation density up to 104 spores per plate; higher densities strongly inhibit macrophorogenesis and stimulate microphorogenesis (Fig. 1). The number of microphores per inoculated spore varies from several thousand (with 10 spores per plate) to less than one (with 10 7 spores per plate). Plates inoculated with 10 spores produce about 120 macrophores per spore, and those inoculated with 10 7 spores produce none. 'Sealing' (placing two cultures in a tight 2.4-1 box) stimulates microphorogenesis and inhibits macrophorogenesis (Fig. 1). Two cultures placed in a tight 14-1 box behave like 'unsealed' cultures. For standard conditions we adopted unsealed cultures inoculated with 105 spores per plate. Cultures inoculated with 105 spores and incubated in the dark exhibit a remarkable degree of synchronization in sporangiophore development (Fig. 2). In the first 2 d there are no sporangiophores; maximal values for both microphore numbers and macrophore dry weight are found 4 d after inoculation and maintained for at least 6 d more. Therefore, in all other experiments, phorogenesis was determined after a 4-d incubation of the cultures. The macrophores formed at low plating densities (about 10 spores per plate) carry large macrosporangia with some 10 s spores each. Under our

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standard conditions (10 5 spores per plate), the macrosporangia are much smaller and contain only about 1000 spores each. The microsporangia at the tips of microphores also contain about 1 000 spores each (Guti6rrez-Corona and Cerd~Olmedo 1985). The nitrogen supply plays a critical role in phorogenesis (Fig. 3). Microphorogenesis is inhibited at asparagine concentrations over 30 mM. Macrophorogenesis is maximal at about 25 mM asparagine and smaller at higher concentrations under the normal 'unsealed' conditions. This decrease is not found in 'open' plates (incubated without lids in a 14-1 container). The asparagine concentration used in the standard minimal medi-

L.M. Corrochano and E. Cerdfi-Olmedo : Morphogenesis in Phycomyces I

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Fig. 2. Time course of phorogenesis in Phycomyces. Microphores and macrophores were determined in unsealed cultures inoculated with 105 spores and incubated in the dark

Table I. Phorogenesis in cultures of Phycomyces btakesleeanus grown for 4 d in the light or in the dark. Results from 11 independent experiments with two replicas each

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Photophorogenesis. Light inhibits microphorogenesis and stimulates macrophorogenesis (Table 1). Analysis of variance (Sokal and R o h l f 1969) leads to the conclusion that most of the rather large variability of the results occurs between independent

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Fig. 3. Phorogenesis in Phycomyces as a function of the concentration of L-asparagine in unsealed (o) and open (Q) cultures incubated in the dark. The standard asparagine concentration (13.3 raM) is shown by the discontinuous line

experiments and only a comparatively minor fraction (17-27%) between separate replicas of the same experiment. It is therefore preferable to carry out independent repetitions of the experiments, rather than to increase the number of plates within each experiment.

L.M. Corrochano and E. Cerd~t-Olmedo: Morphogenesis in Phycomyces

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Permanent illumination is not essential to inhibit microphorogenesis and stimulate macrophorogenesis. The photosensitive period was determined by dark-to-light and light-to-dark transfer experiments (Fig. 4). Microphorogenesis is affected by light given during any part of the sensitive period, from 32 to 68 h (counted from the moment of inoculation), and only then. Photostimulation of mac-

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L.M. Corrochano and E. Cerdfi-Olmedo : M orphogenesis in Phycomyces

rophorogenesis appears to have a similar time course. Table 2 confirms the high photosensitivity of cultures aged 32 to 68 h. The photosensitive period was further defined by 4-h exposures to light of cultures otherwise incubated in the dark (Fig. 5). The maximum photosensitivity occurs in the cultures aged 44 and 48 h. Discussion

Sporangiophore development in Phycomyces depends strongly on illumination and culture conditions. Our results define a new protocol for the study of photomorphogenesis, a protocol which allows fast and vigorous growth of the cultures and sufficient reproducibility of the results. We recommend the inoculation of 105 viable, heat-activated spores into 25 ml minimal medium in each standard Petri plate and the incubation of the plates at 22 ~ C unpiled and unsealed. We measure two variables, micro- and macrophorogenesis, expressed as absolute number and dry weight, respectively. The two variables usually run in opposite directions, but there is no fixed rate of exchange between them: a macrophore does not substitute for a certain number of microphores. This is particularly clear in the results of Fig. 3 and 4. We thus end up with two distinct light-dependent phenomena. It is not surprising, from an adaptive point of view, that darkness, crowding, lack of ventilation, and scarcity of nitrogen favor microphorogenesis. Under our experimental conditions, macro- and microsporangia contain about the same number of spores, but macrophore construction requires a much larger expenditure of materials and energy. Phycomyces apparently takes light and ventilation as signals that the complex guidance mechanisms of the macrophores would have a chance to bring the spores into the open. In the absence of such signals, microphorogenesis is favoured. The morphogenetic effect of each variable depends on the values of the other variables. Thus, sealing exerts a decisive influence in the presence of high asparagine concentrations. This is understandable if sealing causes the accumulation of a volatile metabolite abundantly excreted by thick mycelia grown on rich media (Russo 1977; Galland and Russo 1979). Our work unveils no differences between the photosystems responsible for micro- and macrophorogenesis, but hints at the existence of the double photosystem for macrophorogenesis suggested by Thornton (1975). According to this view, light

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induces both the formation of macroprimordia (the starting structures of macrophores) and the differentiation into macrophores of some of the microprimordia formed in darkness. Thornton's conclusions would explain the fact that macrophorogenesis is maximal in mycelia transferred to light after about 2 d incubation in darkness (Fig. 4). Photosensitivity is itself a developmental phenomenon with a precise time slot. Cultures are photosensitive both for micro- and macrophorogenesis 32 to 68 h after inoculation, just before the appearance of sporangiophores in the dark controls. The 10 s mycelia in each culture are not precisely synchronyzed. Four-hour illumination at the optimal moment (44-52 h) induces a 701% reduction in the number of microphores relative to the dark controls (Fig. 5). Since the light-fluence used in Fig. 5 is saturating (Corrochano et al. 1988), about 30% of the mycelia in the culture are insensitive to light at that time. These insensitive mycelia had already been sensitive to light. This is shown by the fact that illumination at 48-52 h has the same effect as illumination from 48 h on (compare Figs. 4 and 5). The great photosensitivity of 48 h old cultures allows the use of pulse illuminations of different fluences and wavelengths, appropriate for the determination of action spectra (Corrochano et al. 1988) and other purposes. We thank A. Fernfindez Estefane, J. C6rdoba L6pez, and Dr. Javier Avalos for technical services, Professor J. Lozano Campoy for equipment, and Professor E.D. Lipson for critical reading of the manuscript. This work was supported by Comisi6n Asesora para Investigaci6n Cientifica y Tbcnica and Plan de Formaci6n de Personal Investigador.

References Bergman, K. (1972) Blue-light control of sporangiophore initiation in Phycomyces. Planta 107, 53 67 Cerdfi-Olmedo, E. (1987) Standard growth conditions and variations. In: Phyeomyces, pp. 337 339, Cerdfi-Olmedo, E., Lipson, E.D., eds. Cold Spring Harbor Laboratory, New York Corrochano, L.M., Galland, P., Lipson, E.D., Cerd~-Olmedo, E. (1988) Photomorphogenesis in Phycomyces: Fluence-response curves and action spectra. Planta 174, 315-320 Galland, P., Lipson, E.D. (1987) Light calibrations and radiometric units. In: Phycomyces, pp 375-380, Cerdfi-Olmedo, E., Lipson, E.D., eds. Cold Spring Harbor Laboratory, New York Galland, P., Russo, V.E.A. (1979) The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus. Planta 146, 257-262 Gruen, H.E. (1959) Growth and development of isolated Phycomyces sporangiophores. Plant Physiol. 34, 158-168

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Guti6rrez-Corona, F., Cerdfi-Olmedo, E. (1985) Environmental influences in the development of Phycomyces sporangiophores. Exp. Mycol. 9, 56-63 L6pez-Diaz, I., Cerd~t-Olmedo, E. (1981) Light controlled phorogenesis and mycelial growth in Phycomyces mutants. Curt. Genetics 3, 23-26 Ortiz-Castellanos, M.L., Guti6rrez-Corona, J.F. (1988) The sensitive period for light and temperature regulation of sporangiophore development in Phycomyees. Planta 174, 305-308 Rudolph, H. (1958) Entwicklungsphysiologische Untersuchungen an den Sporangiophoren yon Phycomyces blakesleeanus. Biol. Zentralbl. 77, 385-437 Russo, V.E.A. (1977) The role of blue light in synchronization of growth and inhibition of differentiation of stage I sporan-

giophore of Phycomyces blakesleeanus. Plant Sci. Lett. 10, 373 380 Sandmann, G., Hilgenberg, W. (1979) Der lichtabhfingige Intermedi/irstoffwechsel von Phycomyces blakesleeanus Bgff. III. Der Einflug von Asparagin auf die Bildung der Sporangiophoren. Biochem. Physiol. Pflanz. 174, 794-801 Sokal, R.R., Rohlf, F.J. (1969) Biometry. W. H. Freeman and Co., San Francisco Thornton, R.M. (1972) Alternative fruiting pathways in Phyeomyces. Plant Physiol. 49, 194-197 Thornton, R.M. (1973) New photoresponses of Phycomyces. Plant Physiol. 51,570-576 Thornton, R.M. (1975) Photoresponses of Phycomyces blakesleeanus: initiation and development of sporangiophore primordia. Am. J. Bot. 62, 370-378