Photomorphogenesis in Phycomyces: Competence period and stimulus-response relationships

Journal o f Photochemistry and Photobiology, B: Biology, 5 (1990) 255 - 266

255

PHOTOMORPHOGENESIS IN Phycomyces: COMPETENCE PERIOD AND STIMULUSRESPONSE RELATIONSHIPS L. M. CORROCHANO 5" and E. CERD-k-OLMEDO tf"

Departamento de Gendtica y Biotecnia, Universidad de Sevilla, Apartado 1095, E -41080 Se villa (Spain)

(Received May 29, 1989; accepted August 22, 1989)

Keywords. Blue light, photomorphogenesis, development, Phycomyces blakesleeanus.

Summary The illumination of Phycomyces mycelia (age, 48 h) with blue-light pulses inhibits the future development of dwarf sporangiophores and stimulates the development of giant sporangiophores. Each of these two responses is the sum of two components; the first components of each response have thresholds at about 10 -4 J m -2 and saturate at about 10 -2 J m -2, and the second components reach threshold and saturation at about 1 and 102 J m -2 respectively. Each of the two components of the two responses follows a sigmoidal function of the form ax/(b + x) where x is the light exposure. The function for photomicrophorogenesis may be explained by a proportionality between the response and the concentration of a chemical with the assumption t h a t fight destroys this chemical when it forms a dimer. A slightly modified explanation serves for photomacrophorogenesis. Under our experimental conditions, each culture contains about 10 s separate mycelia. The maximal responses are found when a culture is exposed to a light pulse at the age of 48 h; however, the cultures exhibit some competence to respond to light over a period of about 35 h. The competence periods of individual mycelia in the cultures may be determined indirectly; they are about 6 h on average, but often much less. The competence periods for the two components of the same individual mycelium and of different mycelia in the cultures do n o t always coincide. When the first c o m p o n e n t is active, its saturation blocks all microphorogenesis, so t h a t the second c o m p o n e n t can only be detected in mycelia whose first comp o n e n t is inactive at the time of illumination.

tPresent address: MRC Molecular Genetics Unit, Hills Road, Cambridge CB2 2QH, U.K. t t A u t h o r to whom correspondence should be addressed. Elsevier Sequoia/Printed in The Netherlands

256 The inhibition of microphorogenesis by continuous illumination exhibits a single component, with a threshold at about 10 -s W m -2. This roughly coincides with the expected response of the first component to illumination for the entire competence period of 6 h. The stimulation of macrophorogenesis by continuous illumination exhibits a complicated dependence on light intensity that suggests multiple origins for the giant sporangiophores.

1. Introduction Blue light causes the fungus Phycomyces blakesleeanus to increase its carotene biosynthesis (photocarotenogenesis), to modify its fruiting pattern (photomorphogenesis) and to change the velocity (photomecism) and the direction (phototropism) of sporangiophore growth (reviews in ref. 1). Phycomyces responds to a very broad stimulus range, from very dim to very bright light. To make this possible, each photoresponse is the sum of two separate components with different thresholds and saturating intensity levels, as shown for photocarotenogenesis [2, 3], phototropism [4], lightinduced absorbance changes [5] and photomorphogenesis [6]. There are two photomorphogenic responses in Phycomyces: photomacrophorogenesis, the induction of giant sporangiophores, and photomicrophorogenesis, the inhibition of dwarf sporangiophores (reviews in refs. 7 and 8). A new method for studying these responses has led to the observation that only mycelia of a certain age are competent to respond to light [9] and detailed action spectra have been recorded [6]. This paper reports the effects of pulsed and continuous illumination on photomorphogenesis in Phycomyces; the periods of competence in which large populations are able to react to light are defined and the competence periods of individual mycelia are calculated.

2. Experimental details

2.1. General procedure Strain, growth conditions, measurement of light and assays of morphogenesis have been described previously [9]. The general procedures for culture, handling and illumination of Phycomyces are described in ref. 1. Each plate was inoculated with 10 s heat-activated spores and incubated for 4 days at 22 + 1 °C in the dark before counting the microphores and plucking, drying and weighing the macrophores. For pulsed blue illumination, plates were placed in a black cardboard box. Blue light was provided by a quartz halogen lamp (Sylvania FCS, 150 W) installed in a slide projector (Hanimex 2100 EF) with two heat filters (KG-1; Schott, Mainz, F.R.G.), the appropriate neutral density filters (type

257 NG; Schott) and a broad-band blue filter (5-61; Coming Glass Works, Coming, NY, U.S.A.) with a maximal transmission at 440 nm. The source for continuous illumination at intensities greater than 10 -6 W m -2 was a set of five fluorescent lamps (Sylvania F15T8/CW) and for lower intensities three incandescent lamps (25 W, Tensor Corp., Brooklyn, NY, U.S.A.) connected to a variable transformer were used. The t w o sources gave similar results around 10 -6 W m -2. Two plates at a time were incubated in a light-tight w o o d e n b o x of capacity 14 1 one side of which was made of blue plastic (type 2424, RShm and Haas, Philadelphia, PA, U.S.A.; maximal transmission at 465 nm) with a variable number of neutral density plastic sheets (Plexigias G, RShm and Haas) to adjust the blue-light intensity. Red light was filtered from the fluorescent lamps with three red plastic sheets (type S10218, Mitsubishi R a y o n Co.). 2.2. Data analysis

The experimental results were fitted [6] by an error-weighted nonlinear least-squares m e t h o d to the equation y = 1 + a x / ( x + b) + c x / ( x + d )

(1)

where x is the exposure in joules per square metre, y is the observed response (microphorogenesis or macrophorogenesis) and a, b, c and d are four adjustable parameters. A normalized version of the results from this paper and from ref. 6 was obtained using the equations y' = (y -- 1)/a

x' = x/b

y' = (y -- 1 -- a)/c

x' = x/d

(2) (3)

which were applied to the first and second components respectively (see Fig. 6).

3. Results and discussion 3.1. M o r p h o g e n i c r e s p o n s e s t o b l u e l i g h t

Under our experimental conditions, cultures grown in the dark contain both microphores and macrophores. After 4 days each plate contains 1.17 × l 0 s+ 2.5 × 103 microphores and the dry weight of the macrophores is 108 + 8 mg (mean and its standard error in ten independent experiments). Broadband blue-light pulses given to mycelia (age, 48 h) inhibit microphorogenesis and stimulate macrophorogenesis (Fig. 1). The mycelia produce macrophores and microphores in numbers determined by the illumination. Each morphogenetic response is the sum of two sigmoidal components with different thresholds and amplitudes. The threshold of the low-exposure c o m p o n e n t is a b o u t 10 -4 J m -2 and that of the high-exposure c o m p o n e n t is a b o u t 1 J m -2 for both microphorogenesis and macrophorogenesis.

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Fig. 1. Morphogenesis after blue-light pulses. The number of microphores and the dry weight of macrophores in cultures (age, 4 days) exposed to blue light for 2 rain at the age of 2 days relative to the values found in cultures kept continuously in the dark. Each point represents the mean in ten independent experiments and the bars represent the standard error of the mean when larger than the size of the points. To give an exposure of 104 J m -2, an illumination time of 20 rain was used. The continuous lines represent the computer-fitted algebraic functions described in the text.

As n o t e d f o r m o n o c h r o m a t i c light [ 6 ] , e a c h sigmoidal c o m p o n e n t can b e r e p r e s e n t e d b y an e x p r e s s i o n o f t h e t y p e y = ax/(x

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w h e r e x is t h e e x p o s u r e ; y is t h e r e s p o n s e ; a is t h e a m p l i t u d e o f t h e r e s p o n s e a n d b is t h e e x p o s u r e a t t h e i n f l e c t i o n p o i n t o f t h e sigmoid. In this f u n c t i o n , t h e e x p o s u r e giving 91% o f t h e r e s p o n s e is 10b a n d t h a t giving 9% o f t h e r e s p o n s e is b / l O . T h u s 82% o f t h e r e s p o n s e occurs w i t h i n a 1 0 0 - f o l d range o f e x p o s u r e values or t w o d e c a d e s in t h e l o g a r i t h m i c p l o t . T h e r e s p o n s e s in Fig. 1 fit t h e e x p r e s s i o n s m = 1 -- 0.37x/(x

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w h e r e x is t h e e x p o s u r e (J m -z) a n d m a n d M are t h e n u m b e r o f m i c r o p h o r e s a n d t h e d r y w e i g h t o f m a c r o p h o r e s r e s p e c t i v e l y in i l l u m i n a t e d cultures relative t o t h o s e f o u n d in t h e d a r k . T h e p a r a m e t e r s o f these e x p r e s s i o n s w e r e o b t a i n e d f r o m least-squares fits t o t h e e x p e r i m e n t a l results. T h e d u r a t i o n o f t h e e x p o s u r e d o e s n o t p l a y a critical role in t h e r e s p o n s e . T h e B u n s e n R o s c o e r e c i p r o c i t y law is valid, at least f o r e x p o s u r e s b e t w e e n 12 s a n d 20 m i n [ 6 ] . Figure 2 e x t e n d s r e c i p r o c i t y t o 2 0 0 m i n f o r b o t h c o m p o n e n t s o f b o t h responses.

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Fig. 2. Morphogenesis after blue-light pulses. The number of microphores and the dry weight of macrophores in cultures (age, 4 days) exposed to blue light for 2 rain (open circles) or 200 rain (filled circles) at the age of 2 days relative to the values found in c u l t u r e s k e p t c o n t i n u o u s l y in t h e dark. E a c h p o i n t r e p r e s e n t s t n e m e a n in t h r e e i n d e p e n d e n t e x p e r i m e n t s a n d t h e bars r e p r e s e n t t h e s t a n d a r d e r r o r o f t h e m e a n w h e n larger t h a n the size o f t h e p o i n t s . T h e c o n t i n u o u s lines r e p r e s e n t t h e algebraic f u n c t i o n s f r o m Fig. 1. T h e e x p e r i m e n t a l t h r e s h o l d s w i t h pulses o f 2 a n d 2 0 0 m i n are n o t statistically d i f f e r e n t .

3.2. Competence periods for photomicrophorogenesis Competence to detect blue light and to respond to it is a developmental p h e n o m e n o n (Fig. 3). Mycelia of different ages were exposed to blue-light pulses of 0.34 J m-2; these pulses saturate the first c o m p o n e n t of the responses b u t do n o t induce the second component. Maximum competence occurs in mycelia aged 48 h and some response is found in mycelia aged 36 68 h. Thus the overall competence period lasts a b o u t 32 h. Larger responses are obtained with brighter pulses (i.e. 350 J m -2) which saturate both components, b u t evolution with time and the competence period remain approximately the same. The peak responses coincide with the saturating responses in Fig. 1 as expected. The subject of these experiments is a large population of a b o u t l 0 s individual mycelia grown together in a Petri dish. They may n o t all be competent at the same time. This means that not even the brightest light pulses destroy microphorogenesis totally (Figs. 1 - 3 and eqn. (4)). At the age of 48 h, only 37% of the mycelia are c o m p e t e n t to induce the low-exposure comp o n e n t of the response, 19% are c o m p e t e n t to induce the high-exposure component, b u t n o t the low-exposure component, and 44% cannot respond at all because they are incompetent for both. According to this interpretation, the response of a Petri dish culture of a given age to a saturating exposure is equal to the proportion of individual mycelia that are c o m p e t e n t to respond. If each mycelium remains c o m p e t e n t for tc h and the Petri dish is repeatedly exposed to light at intervals of tc h,

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then each mycelium will be stimulated once and all mycelia will respond. If different Petri dishes are exposed at different ages, separated by intervals of tc h, the sum of the responses in all the cultures will be unity. If the interval between the exposures is ti, the sum of the responses in all the cultures will be tc/ti. For example, if the competence period t¢ is 8 h and the interval ti between exposures of different plates is 4 h, then homologous mycelia on different plates (e.g. those whose competence begins at the age of 39 h and ends at the age of 47 h will be stimulated in two of the cultures (those illuminated at the ages of 40 and 44 h) and the sum of the responses in all plates will be two. In Fig. 3, t i is 4 h and the sum of the first-component responses is 1.52 (the area between the open circles, ages 36 - 68 h, and the horizontal line of ordinate unity). The competence period of the first component may then be estimated as t c = 1.52 X 4, or 6.1 h. The sum of the second-component responses is 2.97, but this includes the low-intensity responses; the specific second-component response is the difference, 1.45, and the competence period is 5.8 h. This interpretation implies that individual mycelia are competent for a much shorter period than the population as a whole. In the population, the competence periods for the first and second components coincide. In each individual mycelium, the competence periods for the first and second components do not always coincide. If they did, the first component, when

261

saturated, would abolish all microphorogenesis and the second c o m p o n e n t would be undetectable. The results of multiple exposures (Table 1) support this interpretation of microphorogenesis. Repeated exposure of a population at different times causes a larger reduction in microphore count than a single exposure at the m o m e n t of maximal competence. This is true for exposures that saturate one or both components of the response. The results in Table 1 indicate that the competence periods of many mycelia last less than 4 h, since a considerable number of microphores (23% of the dark control) are seen in cultures briefly illuminated every 4 h.

3.3. Microphorogenesis under continuous illumination Our interpretation predicts that very long exposures should affect more mycelia than shorter exposures and should reduce the microphore counts. In fact, continuous illumination for 12 h (age 36 - 48 h) reduces the number of microphores to 6.6% of the dark control. This indicates considerable synchrony in the development of competence, since about 93% of the mycelia must be competent during the indicated period of 12 h. There are no traces of two separate components under continuous blue illumination (Fig. 4). As expected from action spectra [6], red light has no effect on morphogenesis, and may be used as a safe-light for manipulations that must be carried out in the dark.

TABLE 1 M o r p h o g e n e s i s a f t e r m u l t i p l e blue-light pulses. T h e n u m b e r o f m i c r o p h o r e s a n d t h e d r y w e i g h t o f m a c r o p h o r e s in c u l t u r e s (age, 4 days) e x p o s e d t o blue l i g h t at t h e age o f 2 days relative t o t h e values f o u n d in c u l t u r e s k e p t c o n t i n u o u s l y in t h e dark. ( T h e m e a n a n d its s t a n d a r d e r r o r f r o m t h r e e i n d e p e n d e n t e x p e r i m e n t s are given)

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Fig. 4. Morphogenesis in cultures grown continuously under blue light. The number of microphores and the dry weight of macrophores in cultures (age, 4 days) grown in the light relative to the values found in cultures kept in the dark. Each point represents the mean in four to six independent experiments and the bars represent the standard error of the mean when larger than the size of the points. The triangles indicate results obtained with red light. The results with continuous blue light may be at t ri but ed to the operation of the first c o m p o n e n t during the c o m p e t e n c e period. Continous illumination gives all mycelia a chance to respond. Saturation of the first c o m p o n e n t totally abolishes microphorogenesis and the operation of the second c o m p o n e n t c a n n o t be observed. A very simplified view of the effects of continuous illumination is that t h ey are equivalent to those o f pulses of 6 h covering the c o m p e t e n c e period o f each mycelium. Using this assumption, the results of Figs. 1 and 4 can be quantitatively com par e d by making the exposures in Fig. 1 equal to the p r o d u c t o f 21 600 s (6 h) and t o the intensities in Fig. 4. The results of continuous illumination then fall within less than a logarithmic decade of those o f the first c o m p o n e n t of pulsed i l l u m i n a t i o n - - n o t t o o large a difference given the enor m ous stimulus intervals under study. Figure 5 confirms t hat illumination outside the c o m p e t e n c e period has no effect; it does n o t even m o d i f y the sensitivity o f the mycelium at later times.

3.4. Comparison o f the two morphogenic responses The ex p o s ur e- r es pons e curves of microphorogenesis and macrophorogenesis have the same inflection points, and therefore the same threshold and

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saturating exposures, once the standard errors of the determinations are taken into account. The inflection points of eqn. (5) are 0.0016 + 0.00092 and 11.2 + 1.4; those of eqn. (6) are 0.0022 + 0.0020 and 8.0 + 5.2. The determinations of the competence periods of macrophorogenesis (Fig. 3) are less clear than those for microphorogenesis, b u t point in the same direction. The stimulation of macrophorogenesis by continuous illumination exhibits a more complex dependence on light intensity than the inhibition of microphorogenesis (Fig. 4). For continuous exposures to intensities greater than 10 -3 W m -2, macrophorogenesis decreases as the intensity increases. This surprising behaviour may be explained by the observations of Thornton [10]. Mycelia growing in the dark develop both macroprimordia and microprimordia, which later develop into macrophores and microphores respectively. Light stimulates the development of macroprimordia, inhibits that of some microprimordia, and converts other microprimordia into macroprimordia. The increase in macrophores is due, in part, to an increased number of macroprimordia and, in part, to the formation of macrophores in some microprimordia. High intensities inhibit the production of micro-

264

primordia and block one of the components of this sum, leading to decreased macrophorogenesis. 3.5. S i g m o i d a l e x p o s u r e - r e s p o n s e r e l a t i o n s h i p

Figure 6 shows the fit of all the experimental results that were subject to mathematical analysis using the sigmoidal function defined by eqn. (4). This is a second-order function, n o t unlike those found for various chemical and biological phenomena, such as DNA hybridization kinetics and enzyme activity. In the normalized equation for photomicrophorogenesis, m = 1 x / ( x + b), x is the exposure, i.e. the product of the light intensity I and the exposure time t. Taking the derivative of m with respect to t, we obtain d m = - - b - l m 2 I d t . This second-order equation may be explained in molecular terms by assuming that microphorogenesis m is proportional to the concentration of a chemical and that this chemical is destroyed by light in proportion to its intensity and to the square of the concentration of the chemical. This suggests that the chemical has the ability to form dimers and that only dimers are destroyed by light. For the stimulation of macrophorogenesis b e y o n d its level in the dark, M = a x / ( x + b) a n d d M = (ab) -1 ( M - - a)2Idt. This second-order equation may be explained in molecular terms by assuming that the photo-induced production of macrophores is proportional to the concentration of a chemical and that this chemical is generated by light in proportion to its intensity and to the square of the concentration of the chemical minus a constant amount of the chemical (possibly because this constant amount

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265

does not undergo dimerization). This result differs sufficiently from that of microphorogenesis to require different molecular mechanisms for the two responses. The two components of each response follow the same equations and can therefore be explained in identical terms. The 104-fold variation in sensitivity between the two components may be due to differences in the concentration or the activity of the photoreceptor or other elements of the transduction chain. The slight differences in the action spectra of the two components [6] suggest that the corresponding photoreceptors are not identical. The working model of photomorphogenesis which has been derived by fitting algebraic equations to the physiological results may now be tested in different ways, most notably by using available mutants and by the isolation of new mutants.

Acknowledgments The continuous illumination experiments were carried out during a visit of L. M. C. to Syracuse University (supported by a grant from the U.S.Spain Joint Committee for Scientific and Technological Cooperation). We thank Prof. Edward D. Lipson and Dr. Paul Galland for their hospitality and help, A. Fernandez Estefane, J. CSrdoba LSpez and Dr. Javier Avalos for assistance, Prof. J. Lozano Campoy for equipment and the ComisiSn Interministerial de Ciencia y Tecnolog/a, Madrid, for financial support (BT8734).

References 1 E. Cerd~-Olmedo and E. D. Lipson (eds.), Phycomyces, Cold Spring Harbor Laboratory, New York, 1987. 2 M. Jayaram, D. Presti and M. Delbriick, Light-induced carotene synthesis in Phycomyces, Exp. Mycol., 3 (1979) 42 - 52. 3 E. R. Bejarano, J. Avalos, E. D. Lipson and E. Cerd~-Olmedo, Photoinduced accumulation of carotene in Phycomyces, Planta, submitted for publication. 4 P. Galland and V. E. A. Russo, Light and dark adaptation in Phycomyces phototropism, J. Gen. Physiol.. 84 (1984) 101 - 118. 5 C. H. Trad and E. D. Lipson, Biphasic fluence-response curves and derived action spectra for light-induced absorbance changes in Phycomyces mycelium, J. Photochem. Photobiol. B, 1 (1987) 305 - 313. 6 L. M. Corrochano, P. Galland, E. D. Lipson and E. Cerd~-Olmedo, Photomorphogenesis in Phycomyces; fluence-response curves and action spectra, Planta, 174 (1988) 3 1 5 - 320. 7 V. E. A. Russo, P. Galland, M. Toselli and L. Volpi, Blue light induced differentiation in Phycomyces blakesleeanus. In H. Senger (ed.), The Blue Light Syndrome, Springer, Berlin, 1980, pp. 563 - 569. 8 P. Galland and T. Ootaki, Differentiation and cytology, in E. Cerd~-Olmedo and E. D. Lipson (eds.), Phycomyces, Cold Spring Harbor Laboratory, New York, 1987, pp. 281 - 316.

266 9 L. M. Corrochano and E. Cerd~-Olmedo, Photomorphogenesis in Phycomyces: dependence on environmental conditions, Planta, 174 (1988) 309 - 314. 10 R. M. Thornton, Photoresponses of Phycomyces blakesleeanus: initiation a development of sporangiophore primordia, A m . J. Bot., 62 (1975) 370 - 378.