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Phosphatase Elements
Fungal Genetics and Biology 28, 201–213 (1999) Article ID fgbi.1999.1175, available online at http://www.idealibrary.com on
Blue Light Signaling Chains in Phycomyces: Phototransduction of Carotenogenesis and Morphogenesis Involves Distinct Protein Kinase/Phosphatase Elements
George Tsolakis,* Eleni Parashi,* Paul Galland,† and Kiriakos Kotzabasis*,1 *Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Crete, Greece; and †Fachbereich Biologie, Philipps-Universita¨t Marburg, Lahnberge, 35032 Marburg, Germany
Accepted for publication November 9, 1999
The reversible, dynamic phosphorylation of Ser, Thr, Tyr, or His residues of key proteins is a widely utilized mechanism for regulating a plethora of cellular events. Processes that are tightly controlled by protein phosphorylation require the catalytic activity of specific protein kinase(s) (PrK) for the addition of phosphate and specific protein phosphatase(s) (PrP) for the removal of phosphate. In principle, the steady-state level of phosphate in a target protein substrate can be modulated by the activities of the cognate PrK or PrP or both. The balance of these enzymatic activities defines in turn the strength and duration of the physiological event (Hunter, 1995). Protein phosphorylation as a regulatory mechanism can ensure signal relaying and amplification as well as signal(s) integration (e.g., the mitogen-activated protein kinase module; Ferrell, 1996); moreover, it can also offer efficient deactivation by switching the signal(s) off [e.g., the phototransduction cascade of vertebrates/invertebrates (Scott and Zucker, 1997)]. During the past decade, exciting studies implicating protein phosphorylation in blue light (BL)2-regulated phenomena of plants and fungi have been conducted. Among them, phototropism has gained the most attention. In several mono- and dicotyledonous plants, the BL-dependent protein phosphorylation of a plasma membrane protein of about 120 kDa in vivo and in vitro is well
Tsolakis, G., Parashi, E., Galland, P., and Kotzabasis, K. 1999. Blue light signaling chains in Phycomyces: phototransduction of carotenogenesis and morphogenesis involves distinct protein kinase/phosphatase elements. Fungal Genetics and Biology 28, 201–213. Carotenogenesis and morphogenesis represent two of the several responses sensitive to blue light which characterize the lower eukaryote Phycomyces blakesleeanus. Speculating that reversible phosphorylation may be an intracellular event beyond the photoperception step, we resorted to the use of first-choice inhibitors of protein phosphatases and protein kinases. The mycelial -carotene content of dark-grown cultures was induced by all agents administered, while the morphogenic output showed the typical trend effected by light only with one of the protein kinase inhibitors. Our data provide convincing evidence that protein phosphorylation plays a regulatory role in photocarotenogenesis and photomorphogenesis of Phycomyces. According to the model we propose, the putative signaling elements involved are anticipated to have a repressive function in the dark so that the responses are maintained in the ‘‘off ’’ mode until the moment photon information has to flow through the regulatory circuit. r 1999 Academic Press
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To whom correspondence should be addressed. Fax: 0030-81-394408. E-mail: [email protected].
1087-1845/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
Abbreviations used: BL, blue light; OKA, okadaic acid; GEN, genistein; STS, staurosporine.
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established and represents an early tie in the transduction chain for phototropism (reviewed by Short and Briggs, 1994). Evidence in support of this connection first stemmed from in vitro studies with the nonphototropic hypocotyl (nph) mutants of Arabidopsis (Reymond et al., 1992; Liscum and Briggs, 1995). The determination of an asymmetrical microdistribution of the BL-induced protein phosphorylation at the irradiated versus the shaded side of Avena coleoptiles (Salomon et al., 1997) also strongly correlated the signaling aspect of phototropism with the dynamics of protein phosphorylation. Recently, the 120kDa phosphoprotein was suggested to be encoded by the nph1 gene of Arabidopsis, whose predicted amino acid sequence in the carboxy terminus contains motifs typical of a Ser–Thr PrK family within the PrK C group (Huala et al., 1997). In addition, an involvement of PrPs has been demonstrated for light-regulated gene expression in plants. A stimulatory role of PrP1 and PrP2A appears essential in the light-dependent expression of photosynthesis genes of maize (Sheen, 1993). Similarly, in barley, PrP1 and/or PrP2A is required to activate the BL-responsive promoter of the chloroplast psbD gene and the expression of other light-sensitive plastid- or nuclear-encoded genes (Christopher et al., 1997). For the ascomycete Neurospora, roles for PrK and PrP in BL transduction have been reported. Biochemical analyses demonstrating that light affects the phosphorylation states of several mycelial proteins with patterns in the wild type distinct from patterns of the BL-insensitive white collar mutants are notable (Lauter and Russo, 1990; Oda and Hasunuma, 1994). Furthermore, the recently described psp mutant, which completely lacks the ability to phosphorylate a 15-kDa protein as a normal response to BL, has offered a biochemical model system for further molecular analysis (Oda and Hasunuma, 1997). Further evidence of a close link between BL-induced phosphorylation and BL-sensitive physiological processes has been established for microalgae (Ma¨tschke et al., 1997; Pan et al., 1997). Most of the aforementioned studies prompted us to investigate the possible role of protein phosphorylation in two BL-sensitive physiological processes of Phycomyces blakesleeanus, carotenogenesis and morphogenesis. The photosignaling aspect of both responses is well described genetically (reviewed in Corrochano and Cerda-Olmedo, 1992) but the biochemical or molecular nature of the involved components remains unknown.
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We combined the ease in experimental manipulations Phycomyces offers with a pharmacological approach, namely, systematic feeding with widely used cell-permeable inhibitors of the enzymes that catalyze changes in phosphorylation. Our findings provide initial evidence that protein phosphorylation events are part of the network of activities brought into action by BL in this organism.
MATERIALS AND METHODS Chemicals. Okadaic acid (OKA) and okadaol were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA); genistein (GEN) and daidzein were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA); NaF and staurosporine (STS) were from Sigma Chemical Co. (St. Louis, MO). All other chemicals were obtained from Sigma and Merck (Frankfurt, Germany). Organism and culture conditions. All experiments were performed with the standard wild-type strain NRRL1555(-) of P. blakesleeanus on minimal agar plates (8 cm in diameter; Cerda-Olmedo, 1987). For convenience in handling, the solidified content (25 ml) of each dish was covered with circularly cut dialysis membrane (Visking C/110; Serva, Heidelberg, Germany) of the same diameter, which was autoclaved and then rinsed in water/liquid minimal medium. Each plate received 105 heat-activated spores (48°C, 10 min) in water suspension, which were carefully spread onto the membrane disk, according to Corrochano and Cerda-Olmedo (1988). Inoculated dishes were incubated without their lids in foil-wrapped cardboard boxes (four plates per box; the volume of the box with its cover was 14 L). All boxes were kept in a dark, air-conditioned place at 22 ⫾ 1°C. Treatments. Stock solutions of the various inhibitors were prepared in DMSO solution (2.5% v/v for okadaic acid and okadaol or 50% v/v for genistein, daidzein, and staurosporine) and stored as aliquots at ⫺20°C. Sodium fluoride stock solution was prepared in sterile 0.02% v/v Tween 80 water solution and maintained at room temperature. To prepare treatment solutions, all stock aliquots of the appropriate volume were further diluted immediately before use in sterile 0.02% v/v Tween 80 water solution (pH adjusted to 7.0 with a few drops of a 10 N solution of NaOH). Care was taken that the DMSO concentration in the final treatment solution was always adjusted to 0.05% v/v for okadaic acid and okadaol or 1% v/v for the other
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inhibitors in all doses administered and in control treatments (no inhibitor). The administration of a treatment solution took place 43 h after inoculation in a light-tight room under dim red-safe light obtained through a red filter (Plexiglass GS Rot 501, 3 mm thick; Ro¨hm GmbH, Darmstadt, Germany). A 1-ml aliquot of a treatment solution was added to each plate by injection between the membrane disk and the surface of the solid medium. In the light experiments, each cardboard box was uncovered, screened with a blue filter sheet (Plexiglass GS Blau 610, 3 mm thick; Ro¨hm GmbH), and given a 2-min light pulse 5 h after the injection (48th h of the culture) in the same light-tight room. The light source above the blue filter was a 15-W cool-white fluorescent lamp (TL-D, Super 80; Philips, Holland) and the BL energy flux reaching the plates was adjusted in situ to 0.02 J m⫺2 by the use of a photoradiometer system (IL760/IL1700/IL780; International Light, Newburyport, MA). In the dark experiments, culture plates were treated exactly as the BLirradiated plates, except that the light source was not turned on. Measurements of -carotene. The extraction protocol applied in mycelia 72 h after inoculation was essentially as described by (Raugei et al., 1982) with the following modifications: The preweighted mycelial portion of a plate intended for extraction was immersed in 9 ml ice-cold methanol, cut into smaller pieces instead of being powdered in a mortar with liquid nitrogen, and extracted as such. The other portion of the mycelial pad was used for dry weight determination after baking at 110°C for 14 h. All methanolic extractions were performed at 65°C. Morphogenesis assays. Morphogenic assays were carried out as described elsewhere (Corrochano and CerdaOlmedo, 1988). All macrophores from each plate were collected with forceps at the 96th h of cultures and their dry weights measured after drying at 110°C for 14 h. The visible sporangia of microphores were counted on the same macrophore-free plates under a 40⫻ stereomicroscope through calibrated grids set up within the eyepieces. Mean microphore density per square millimeter was calculated from counts of six to eight randomly chosen areas (24–32 quadrats sampling) on the mycelial surface of a plate. Statistical analyses. Treatments were repeated two to four times with two or four plates per experiment. Data from independent experiments were analyzed by the one-way analysis of variance (ANOVA) procedure at ␣ ⫽ 0.05. In cases in which the ANOVA results suggested at least one equality among the means of the treatment
effects, the Fisher multiple comparison procedure (LSD test) was applied among these treatment groups at the ␣ ⫽ 0.05 or 0.01 levels. Results obtained by the Fisher test are given in the figure legends. All analyses were performed with the SYSTAT software (version 5.0; Systat Inc., 1992).
RESULTS AND DISCUSSION Signal transduction studies usually exploit the biochemistry of signaling. Thus, a signaling chain can be made to work or not work by challenging the system under study, in the presence/absence of the primary stimulus, with agents (biochemical analogues, inhibitors, activators) known to influence the putative activity of interest involved in the information flow. A representative breakthrough that ensued from this approach was the identification of signaling intermediates in phytochrome pathways (reviewed by Mustilli and Bowler, 1997). We decided to apply the same experimental rationale to P. blakesleeanus, a lower eukaryote long known mainly for the complex behavior of its sporangiophores (fruiting bodies) in response to blue light (reviewed in Galland, 1990). Two easily assayable photoresponses of this organism were chosen as markers in our experiments: morphogenesis and carotenogenesis. Morphogenesis is a developmental process that can be characterized by the dry weight of macrophores (giant sporangiophores) and the number of microphores (dwarf sporangiophores). Carotenogenesis refers to the mycelial accumulation of -carotene. Illumination, especially BL, increases the macrophore yield and the -carotene content but decreases the production of microphores. The biochemical and molecular properties of the BL signaling network operating in these responses remain elusive. Speculating that reversible phosphorylation may be an intracellular event beyond the primary activation of BL receptors, we present here data from experiments in which inhibitors of PrP and PrK were used. In all treatments reported below, the culture conditions employed were those recommended by Corrochano and Cerda-Olmedo (1988). A main advantage of this surface culture system is that both photomorphogenesis and photocarotenogenesis exhibit simultaneous periods of maximum photocompetence (Bejarano et al., 1990). Therefore, using the same batch of growing culture plates, all responses can be recorded at the proper developmental moment without special requirements for each separate response.
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The Effect of PrP Inhibitors on Dark-Grown Cultures Millimolar concentrations of the widely used PrP inhibitor NaF significantly stimulated the -carotene content of dark-grown mycelia in a dose-dependent manner (Fig. 1). The maximal stimulation was seen between 1 and 40 mM NaF, despite the apparent plateau (⬃60% above dark control values) from 1 to 14 mM and the declining trend above 4 mM. Up to 1 mM NaF, both morphogenic variables were insensitive to it, but above 4 mM, a developmental behavior typical of growth under unfavorable conditions was observed (Corrochano and CerdaOlmedo, 1988): increase in the number of microphores and reduced macrophorogenesis. This behavior should be attributed to fluoride toxicity and should not be considered response-specific since, in the same concentration range, -carotene levels are also declining. According to the general rationale given above, mimicking light action under dark conditions with a chemical of known action against an intracellular target constitutes a
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gross but not sufficient hint that the same type of action on this target is also effected by photons. Thus, BL information in mycelia must be transduced at least via a kind of PrP inhibition for carotenogenesis. This PrP involvement may be an event irrelevant to the chains determining the morphogenic decisions, since the morphogenic output appeared unaltered after the fluoride treatment in the concentration range in which (skoto)carotenogenesis was manifested. The positive effect of fluoride on mycelial carotenogenesis of Phycomyces was first reported by Desai et al. (1973). They correlated the fluoride stimulation in carotene level with a considerable drop in the total activities of acid phosphatase and pyrophosphatase. These phosphatases were thus suggested to be part of the mechanism controlling the biosynthetic flux to -carotene. In a more recent study of alkaline phosphatase from Phycomyces, it was also noted that tetramisole, a potent inhibitor of this enzyme, could increase -carotene of macrophores up to fivefold when included in solid minimal medium (cited in Cohen, 1985).
FIG. 1. Effects of NaF on mycelial -carotene, macrophores dry mass, and microphores numbers of dark-grown cultures. Points and bars (mean ⫾ SE) with common letters at their side define groups of statistical similarity among the corresponding treatments at ␣ ⫽ 0.05 by Fisher’s multiple comparison procedure. The absolute mean values of dark controls that were set as 100% for each variable were -carotene, 72.17 ⫾ 3.15 µg/g of dry mycelial weight; macrophores dry weight, 125 ⫾ 1.108 mg/plate; microphores density, 56 ⫾ 3 mm⫺2.
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Blue Light Signaling in Mycelia of Phycomyces
All the above led us to experiment with OKA, a reagent that does not inhibit acid or alkaline phosphatases (Hardie, 1993). This 38-carbon fatty acid acts as a noncompetitive or mixed inhibitor of serine/threonine PrP1 and PrP2A and is useful because the PrP1/PrP2A sensitivities to OKA have been substantially conserved in most eukaryotic cells (Cohen et al., 1990). The dose–response study of Fig. 2 supports the fluoride data. Up to 0.5 µM OKA, -carotene levels rise over dark control levels up to ⬃70%, while the vein of increase reappeared above a small plateau that extends between 0.5 and 1 µM OKA doses. Both morphogenic variables were insensitive to all okadaic acid concentrations provided, as was the case for NaF. Okadaol, an okadaic acid analogue with approximately 40% of the inhibitory activity of okadaic acid in vitro (Nishiwaki et al., 1990), was effective at 1 µM, increasing -carotene by ⬃25% above dark control values (data not shown). The in vitro IC50 values of PrP2A and PrP1 for OKA
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differ by 3 to 100 times, depending on the cell type, with IC50 values of PrP2A being lower than those of PrP1. Because of this difference, low okadaic acid concentrations are usually expected to inhibit PrP2A selectively in vivo, while a PrP1 inhibition is expected at higher okadaic acid concentrations (Scho¨nthal, 1992). In this respect, a PrP2A/ PrP1 involvement in BL-mediated signaling of carotenogenesis in Phycomyces cannot be ruled out, granted that the participation of these PrPs in cellular regulation is widespread. The present okadaic acid dose–response analyses would be more informative if IC50 values from a Phycomyces cell-free system were available. For this reason, the assignment of selective PrP function to photocarotenogenesis is premature. The notion, however, that two different regulatory activities might be brought into action in a negative way (by their light-mediated inhibition) fits well with the property of photocarotenogenesis being a twostep response with two separate components (Bejarano et al., 1990).
FIG. 2. The inducing effect of OKA on carotenogenesis under dark-growth conditions. Averages accompanied by a letter are significantly different from dark control -carotene levels at the ␣ ⫽ 0.05 level by Fisher’s test. Both morphogenic variables were insensitive to OKA treatment, with ANOVA P values of 0.686 for macrophores and 0.737 for microphores. At all doses plus controls, DMSO was in 0.05% v/v final concentration. Absolute dark control averages: -carotene, 67.053 ⫾ 3.67 µg/g of dry mycelial weight; macrophores dry weight, 116 ⫾ 3.45 mg/dish; microphores counts, 62 ⫾ 2 mm⫺2.
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Analyses with PrK Inhibitors in the Dark The net effect produced intracellularly by an OKA application is a state of increased phosphorylation via the primary inhibition of PrPs and the minor action of the opposing PrKs, provided that they are partially active. Shifting the balance of reversible protein phosphorylation toward a hypophosphorylation state, however, would allow us to see the other side of the picture. To this effect, dark-grown cultures were incubated with PrK inhibitors. STS was first choice because it is a ‘‘catch-all’’ compound inhibiting both serine/threonine- and tyrosine-specific PrKs (Hidaka and Kobayashi, 1993). A dose–response analysis with doses covering three orders of magnitude showed that all three variables under study were susceptible to STS (Fig. 3). At 10 µM, STS was able to induce -carotene synthesis by 50% or increase macrophores dry weight by ⬃35% above the corresponding dark control values. A parallel maximum decrease ⬃60% below dark control
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values was also observed for microphorogenesis at 10 µM STS. The simultaneous STS-evoked variations of microphorogenesis and carotenogenesis, according to the pattern elicited by light, imply that the STS-inhibited target(s) may be, likewise, light-inhibited and a common element in the photocascades operating for these responses. That BLactivated transduction chains in Phycomyces share or are based functionally on common gene products is well established by studies with mutants (Corrochano and Cerda-Olmedo, 1992). Moreover, evidence that heterotrimeric G proteins constitute a common tie in BL signaling circuits regulating morphogenesis, carotenogenesis, and gene expression is strong (G. Tsolakis and K. Kotzabasis, unpublished data). Identification of the intracellular PrK activity(-ies) inhibited by STS is not possible with our in vivo data. In yeasts, however, STS concentrations from 2 to 70 µM have been reported to be sufficient to target
FIG. 3. The effect of staurosporine on carotenogenesis and morphogenesis of dark-grown cultures. Any two averages within each curve that do not have at least one same letter are significantly different at ␣ ⫽ 0.05 by the LSD test. Pairwise comparisons of all points on the macrophores curve showed significant differences at ␣ ⫽ 0.01 level. In all doses plus controls, the final DMSO concentration was 1% v/v. Absolute dark control values were: -carotene, 73.47 ⫾ 4.9 µg/g of dry mycelial weight; macrophores dry mass, 125.5 ⫾ 1.07 mg/dish; microphores counts, 54 ⫾ 2 mm⫺2.
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Blue Light Signaling in Mycelia of Phycomyces
intracellularly the regulatory activity of PrK C, thus implicating its participation in various cell functions (Yoshida et al., 1992; Toda et al., 1993; Branda˜o et al., 1994; Kobori et al., 1994). The only cellular process known to date in Phycomyces that implicates PrK C is spore germination. The role of this signaling regulator was probed by a PrK C-specific activator (a phorbol ester) which prevented the germination process in an early phase after spore activation either by acetate or by heat shock (Carillo-Rayas et al., 1988). Although the authors did not discuss the possibility that a PrK C activity may have to be repressed for the germination to proceed normally after the activating signal, an analogy with the present study—i.e., the use of a PrK inhibitor as a response elicitor—is easily drawn. Notable also is another study wherein STS mimicked in dark-grown cultures of Chlamydomonas the BL-dependent gamete maturation (Pan et al., 1996). In this photosignaling process, an active PrK C-like component was inferred to restore the conversion of pregametes to gametes blocked in the dark. In addition, illumination was suggested to overcome this blockade—probably through an inhibiting effect on the blocking activity—since PrK C-specific activators indeed inhibited gametogenesis in the light. To further evaluate the behavior of STS, genistein, another PrK inhibitor effective against tyrosine-specific PrKs, was utilized. Dark-grown plates given GEN concentrations ranging from 1 to 100 µM showed an increasing trend for mycelial -carotene; maximum levels ⬃70% above the dark control values were obtained at 100 µM GEN (Fig. 4A). The morphogenic expression remained neutral along this treatment range (Fig. 4B). Parallel experiments with daidzein, a negative control compound for genistein (Akiyama et al., 1987), confirmed the specificity of GEN on carotenogenesis and its inability to affect both morphogenic variables (Figs. 4A and 4B). The inducing effect of GEN on carotenogenesis suggests that the light signaling circuit of this response could involve a protein tyrosine kinase additional to those disclosed via OKA or STS. That both PrK/PrP inhibitors are able to promote -carotene levels in the dark indicates indirectly the complexity of the transduction process and, in part, differentiates photomorphogenesis from photocarotenogenesis in the requirement for these putative component(s).
Combinatorial Experiments: An Approach to Order the Effective Components The stimulating effect of the various inhibitors in the dark, described above, suggests that their corresponding
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intracellular targets exist and normally operate under dark conditions, so that mycelial responses are kept ‘‘switched off.’’ These putative signaling components must provide through their catalytic action an overall phosphorylation state for mycelia that is informative of the ambient dark. It seemed reasonable that this default dark-created phosphorylation state might respond either to BL or to the use of PrP/PrK inhibitors. Thus, to test this hypothesis, we included the primary stimulus in the inhibitor treatments (Table 1). An exposure to a nonsaturating BL flux— according to the analyses by Corrochano and CerdaOlmedo (1990)—was chosen with a view to let the responses be expressed quantitatively, avoiding any masking effects caused by light alone. The inhibitor doses tried were those that produced near maximal (okadaic acid, okadaol) or maximal (the other agents) dark output, as determined above. Okadaic acid or genistein in combination with BL (treatments iii and vi of Table 1) turned out to be the most effective treatments in the induction of -carotene (40– 55% above the light control, treatment i of Table 1). The -carotene output from the rest of inhibitors plus BL (treatments ii, iv, and v in Table 1) was weaker, about half of that produced by GEN or OKA plus BL, but also differed significantly from that of the light control (20–26% above light control levels). Concerning photomorphogenesis, only staurosporine (treatment v in Table 1) of all the inhibitors appeared potent enough to increase macrophores significantly (⬃20%) above light control levels or to reduce microphore density by ⬃60% below the corresponding light control output. The synergism between BL and the PrP/PrK inhibitors used discloses that the short light action could be enhanced by a sustained state effected intracellularly via these inhibitors. This enhancement (as measured by the efficiency of the responses) confirms that the intracellular targets of these compounds occupy intermediary positions within the cascades photoregulating the responses under study. Moreover, the observed interaction between BL and inhibitors constitutes another argument in favor of the prediction that photon energy acts negatively on the default PrP/PrK activities to ‘‘switch on’’ the responses. The special sensitivity of morphogenesis to staurosporine as opposed to the indistinct induction of carotenogenesis by all inhibitors, either in the dark or in the light, suggested the experiments of Table 2. Feeding the system with the various inhibitors in combination facilitates the
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FIG. 4. The specific positive effect of genistein (A) on carotenogenesis in dark-grown cultures. This inhibitor treatment had no significant output at the ␣ ⫽ 0.01 level (B) from either macrophorogenesis (ANOVA P ⫽ 0.862) or microphorogenesis (ANOVA P ⫽ 0.431). All variables were unaffected by daidzein (A and B), with nonsignificant differences from dark controls (ANOVA P values: -carotene, 0.991; macrophores, 0.906; microphores, 0.999). Final DMSO concentration and absolute dark control averages as in Fig. 3.
identification of the biochemical entities involved and the development of a hypothetical function scheme. Combining GEN or OKA with STS (treatments vi or vii of Table 2) produced in the dark as much mycelial -carotene as that produced by each of the three inhibitors separately (treatments ii, iii, and iv in Table 2). In contrast,
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the combination of GEN and OKA was particularly productive in -carotene and significantly differed from any other combinatorial or single treatment. Hence, in carotenogenesis, the absence of synergism between STS and GEN or OKA is worthy of note, given that -carotene is induced by all three inhibitors separately. As a possible explanation of
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TABLE 1 Inhibitor Treatments Combined with a BL Pulse Treatment typea i. ii. iii. iv. v. vi. vii.
BL Fluoride (1 mM) ⫹ BL Okadaic acid (1 µM) ⫹ BL Okadaol (1 µM) ⫹ BL Staurosporine (10 µM) ⫹ BL Genistein (100 µM) ⫹ BL Daidzein (100 µM) ⫹ BL
-Carotene
Macrophores
Microphores
100.00b ⫾ 3.870 A 126.75 ⫾ 10.350 B 137.58 ⫾ 6.181 B,C 121.48 ⫾ 6.260 B 124.81 ⫾ 8.100 B 155.30 ⫾ 7.260 C 111.57 ⫾ 3.440 A,B
100.0c ⫾ 1.750A 103.93 ⫾ 1.865 A 100.74 ⫾ 1.730 A 100.41 ⫾ 2.320 A 119.12 ⫾ 1.850 103.50 ⫾ 1.900 A 103.73 ⫾ 1.470 A
100.00d ⫾ 5.151 A 100.05 ⫾ 6.720 A 98.92 ⫾ 4.924 A 102.03 ⫾ 2.850 A 38.30 ⫾ 4.160 99.72 ⫾ 5.290 A 100.38 ⫾ 3.220 A
Note. Dark-grown dishes of Phycomyces blakesleeanus were provided at the 43rd h of culture with 1 ml of the listed reagents and 5 h later were illuminated with a BL flux of 0.02 J m⫺2 for 2 min. Mean ⫾ SE values are expressed in relation to BL control averages which were set at 100%. Significant differences (P ⬍ 0.05) by Fisher’s test are indicated by different capital letters for any two treatment mean values. Absolute dark control averages of all measured variables: 64.63 µg of -carotene/g dry mycelial wt; 0.114 g of dry macrophore mass/plate; 63 microphores/mm2. a Final DMSO concentration 1% v/v in all treatments. b 110.97 µg of -carotene/g dry mycelial wt. c 0.147 g of dry macrophore mass/plate. d 26 microphores/mm2.
the effects of the STS/GEN and STS/OKA treatments on levels of carotenogenesis, the action of one inhibitor seems to mask or block the action of the other. Our statistical analysis, however, cannot discriminate between the action that masks and the action that is masked, but some kind of interaction(s) is hidden. Despite the obscure nature of the interaction, the carotenogenic behavior of the STS/OKA and STS/GEN combinations may be justified. We reason that a masking action could be developed only if the signaling elements targeted simultaneously by STS and OKA or GEN act in the same pathway for controlling carotenogenesis and probably at different regulatory steps.
By analogy, the synergistic interaction between OKA and GEN in carotenogenesis could stem from the action of these agents on signaling components that are not colinear between them but either on the same pathway as the STS-sensitive target. The insensitivity of both morphogenic variables to GEN or OKA is once more confirmed by comparing the pairwise combinations with the STS treatment (treatments v, vi, and vii versus iv in Table 2). If the morphogenic output of the ‘‘light treatments’’ (Table 1) is also taken into account, then the STS-targeted element could be shared by the photosignaling cascade operating for morphogenesis.
TABLE 2 Combinatorial Inhibitor Treatments in Dark-Grown Cultures of Phycomyces blakesleeanus Treatment typea i. ii. iii. iv. v. vi. vii.
Dark control GEN (1 µM) OKA (0.1 µM) STS (5 µM) GEN (1 µM) ⫹ OKA (0.1 µM) GEN (1 µM) ⫹ STS (5 µM) OKA (0.1 µM) ⫹ STS (5 µM)
-Carotene
Macrophores
Microphores
100.00b ⫾ 5.042 150.85 ⫾ 10.400 A 140.22 ⫾ 12.930 A 137.60 ⫾ 5.600 A 177.70 ⫾ 6.348 152.51 ⫾ 8.610 A 148.90 ⫾ 3.960 A
100.00c ⫾ 3.131 A 97.80 ⫾ 2.731 A 100.26 ⫾ 4.800 A 126.00 ⫾ 0.518 B 99.83 ⫾ 0.579 A 125.00 ⫾ 1.150 B 124.00 ⫾ 0.326 B
100.00d ⫾ 3.105 A 99.50 ⫾ 2.700 A 106.95 ⫾ 8.200 A 44.98 ⫾ 0.341 B 97.29 ⫾ 1.280 A 45.43 ⫾ 0.747 B 45.65 ⫾ 0.587 B
Note. Mean ⫾ SE values are shown as percentages of the dark control averages. Statistical similarity grouping among the treatments within each variable according to LSD analyses (P ⬎ 0.05 for carotenogenesis, P ⬎ 0.01 for macro- and microphorogenesis) is defined by common capital letters. a Final DMSO concentration 1% v/v in all treatments. b 64.63 µg of -carotene/g dry mycelial wt. c 0.114 g of dry macrophore mass/plate. d 63 microphores/mm2.
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A Simple Phototransduction Scheme Our data can be most conveniently summarized by arranging the PrP/PrK elements on a branched phototransduction pathway which controls carotenogenesis and morphogenesis (Fig. 5). The so-called pic mutants characterized by a specific defect in the photoinduction of -carotene behave normally with respect to other photoresponses (Lopez-Diaz and Cerda-Olmedo, 1980). In addition, the pim mutants develop microphores even under continuous illumination, which normally inhibits them (Flores et al., 1998). Both these mutants corroborate the notion of a branched photosignaling pathway for carotenogenesis and morphogenesis. Thus, one needs to assume that the pic products act exclusively on the photocarotenogenic branch either before or after the requisite PrTyrK/PrP junction. By analogy, the pim gene products should be located functionally beyond the branchpoint of Fig. 5, exclusively on the morphogenic branch, which remained concealed from our experimental approach. The hierarchy shown in the proposed model, with the STS-sensitive component occurring before the OKA/GEN targets, is not arbitrary. Since the unknown PrK (octagon in
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Fig. 5) via its inhibition by STS potentiates expression of all the response markers for BL signaling, this STS-mediated inhibition should be an early event in the sensory pathway. How close this STS-targeted step lies to the initial event of BL receptor(s) activation(s) cannot be inferred. In the carotenogenic BL-relaying branch, however, by putting the OKA/GEN-sensitive steps downstream of the STSsensitive element, two requirements are met. First, a signaling dissimilarity between carotenogenesis and morphogenesis is obtained according to their differential reactions to the agents used, and second, the hint of the masking relationship discussed above is biased in favor of an OKA or a GEN action working as a mask over the STS action. The synergy between OKA and GEN actions is another theme depicted in the proposed model with two arrows starting from the OKA/GEN-inhibited targets and converging toward the -carotene output. The value of synergistic interactions, at least between signaling transduction pathways, in terms of lending responses greater sensitivity and rapidity has been discussed elsewhere (Fuglevand et al., 1996). Whether the proposed OKA/GENsensitive steps belong to independent photosignaling path-
FIG. 5. A simple model of the various inhibitor treatments and their interactions. The flux through the signaling circuit is given with arrows. The inhibiting action of the exogenous reagents on the proposed targets (polygons) is shown as T lines. T lines and question marks within arrows indicate the possible BL-effected, normal inhibition of the nearby activities. The protein phosphatase (PrP) and protein tyrosine kinase (PrTyrK) polygons are drawn downstream of the protein kinase (PrK) octagon to denote the differential sensitivity of carotenogenesis to OKA and GEN with relation to morphogenesis. The synergy GEN/OKA is indicated with two independent, final arrows converging on the -carotene output. The broken arrows within polygons reflect the proposed default, repressive action of the signaling components to keep the responses in the ‘‘off’’ mode in ambient darkness.
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ways or to one branched pathway cannot be determined. Nonetheless, carotenogenesis is indeed characterized by greater photosensitivity relative to morphogenesis [their lowest thresholds are 10⫺5 and 10⫺4 J m⫺2, respectively (Bejarano et al., 1990; Corrochano and Cerda-Olmedo, 1990)] and is considerably more rapid [e.g., the phytoene dehydrogenase mRNA rises four- to sevenfold in the first 5 min of BL illumination (Ruiz-Hidalgo et al., 1997)]. The putative signaling elements included in Fig. 5 were all identified by inhibitors and this led to the reasoning that BL information may be transduced via these elements by inhibiting their activity. In other words, the action of these transduction components is to help keep the photobehavior ‘‘silent’’ in the dark. Such a type of negative regulation or repressive function in the absence of the light stimulus has been already proposed both for a PrK C-like component in the BL signaling chain operating for the gametic differentiation of Chlamydomonas (Pan et al., 1996) and for the Arabidopsis cop/det/fus mutants which develop light-grown morphologies in complete darkness (Kwok and Deng, 1996). Therefore, consisting not only of positive elements with a downstream activating role but also of negative elements (broken arrows within polygons in Fig. 5) whose light-signal blockade is lifted once the signal flows may be a general feature of aneural photosignaling cascades. The complexity and the interaction types reported here are not unexpected. The genetics of -carotene biosynthesis in Phycomyces has disclosed several gene loci which participate in the regulatory circuit through a variety of interactions, as suggested by numerous studies in mutant strains (Avalos et al., 1992, and references therein; Mehta et al., 1997). On the other hand, the established photosensory pathways of carotenogenesis and morphogenesis are also not lacking in complexity. (The current schemes of the photosensory pathways as defined by genetical data may be found at the following internet address: www.es.embnet.org/ ˜ genus/Phycotrans.html). A branched photocascade for these light responses has also been postulated on the basis of mutant analyses. The products of the genes madA and madB are required both for photocarotenogenesis and for photodifferentiation of sporangiophores (Jayaram et al., 1980; Lopez-Diaz and Cerda-Olmedo, 1980; Corrochano and Cerda-Olmedo, 1990). In the context of our model, these gene products could act either ‘‘upstream’’ or ‘‘downstream’’ of the STS-sensitive PrK. Their signaling position could not, however, be envisaged ‘‘downstream’’ of the proposed branchpoint, because this would mean their exclusive effect on the carotenogenic branch only. Our
model in Fig. 5 is also in accordance with the behavior of other photomutants of Phycomyces, which specifically show defective photocarotenogenesis or photomorphogenesis. That (photo)morphogenesis appeared to be sensitive only to STS does not imply that the intracellular target of this compound is also singular; it also does not imply that the unknown effector target downstream of the STSsensitive step is singular. Neurospora has so far shown that hyphal development normally requires two different PrKs (Yarden, 1992), with one exhibiting a peculiar photoexpression pattern (Lauter et al., 1996) and one PrP (Prokisch et al., 1997). Even if the Neurospora requirements have their counterparts in Phycomyces morphogenic processes, two strategies should be put in future practice. Apart from the development of the proper in vitro cell-free systems in Phycomyces that will identify photoregulated PrK/PrP activities, the use of more specific PrK/PrP substances both in the wild type and in the various mutants will allow us to map the disclosed elements as to the classical gene loci that define the photosensory pathways.
ACKNOWLEDGMENTS We thank Dr. Alexandros Argirokastritis for his useful suggestions and comments on the manuscript.
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212 Cerda´-Olmedo, E. 1987. Standard growth conditions and variations. In Phycomyces (E. Cerda´-Olmedo and E. D. Lipson, Eds.), pp. 337–339. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Christopher, D. A., Xinli, L., Kim, M., and Mullet, J. E. 1997. Involvement of protein kinase and extraplastidic serine/threonine protein phosphatases in signaling pathways regulating plastid transcription and the psbD blue light-responsive promoter in barley. Plant Physiol. 113: 1273–1282. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. 1990. Okadaic acid: A new probe for the study of cellular regulation. Trends Biochem. Sci. 15: 98–102. Cohen, R. J. 1985. Alkaline phosphatase from Phycomyces blakesleeanus. Exp. Mycol. 9: 195–202. Corrochano, L. M., and Cerda´-Olmedo, E. 1988. Photomorphogenesis in Phycomyces: Dependence on environmental conditions. Planta 174: 309–314. Corrochano, L. M., and Cerda´-Olmedo, E. 1990. Photomorphogenesis in Phycomyces: Competence period and stimulus–response relationships. J. Photochem. Photobiol. B:Biol. 5: 255–266. Corrochano, L. M., and Cerda´-Olmedo, E. 1990. Photomorphogenesis in behavioural and colour mutants of Phycomyces. J. Photochem. Photobiol. B: Biol. 6: 325–335. Corrochano, L. M., and Cerda´-Olmedo, E. 1992. Sex, light and carotenes: The development of Phycomyces. Trends Genet. 8: 268–274. Desai, H. G., Desai, J. D., and Modi, V. V. 1973. Regulation of carotenogenesis in Phycomyces blakesleeanus. Indian J. Microbiol. 13: 57–62. Ferrell, J. E. 1996. Tripping the switch fantastic: How a protein kinase cascade can convert graded inputs into switch-like outputs. Trends Biochem. Sci. 21: 460–466. Flores, R., Cerda´-Olmedo, E., and Corrochano, L. M. 1998. Separate sensory pathways for photomorphogenesis in Phycomyces. Photochem. Photobiol. 67: 467–472. Fuglevand, G., Jackson, J. A., and Jenkins, G. I. 1996. UV-B, UV-A, and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell 8: 2347–2357. Galland, P. 1990. Phototropism of the Phycomyces sporangiophore: A comparison with higher plants. Photochem. Photobiol. 52: 233–248. Hardie, D. G. 1993. Use of protein phosphatase inhibitors in intact cells. In Protein Phosphorylation: A Practical Approach (D. G. Hardie, Ed.), pp. 109–119. IRL Press, Oxford. Hidaka, H., and Kobayashi, R. 1993. Use of protein (serine/threonine) kinase activators and inhibitors to study protein phosphorylation in intact cells. In Protein Phosphorylation: A Practical Approach (D. G. Hardie, Ed.), pp. 87–107. IRL Press, Oxford. Huala, E., Oeller, P. W., Liscum, E., Han, I-S., Larsen, E., and Briggs, W. R. 1997. Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science 278: 2120–2123. Hunter, T. 1995. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 80: 225–236. Jayaram, M., Leutwiler, L., and Delbru¨ck, M. 1980. Light induced carotene synthesis in mutants of Phycomyces with abnormal phototropism. Photochem. Photobiol. 32: 241–245. Kobori, H., Toda, T., Yaguchi, H., Toya, M., Yanagida, M., and Osumi, M.
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1994. Fission yeast protein kinase C gene homologues are required for protoplast regeneration: A functional link between cell wall formation and cell shape control. J. Cell Sci. 107: 1131–1136. Kwok, S. F., and Deng, X-W. 1996. The roles of the pleiotropic Arabidopsis COP/DET/FUS genes in repression of photomorphogenic development in darkness. In Regulation of Plant Growth and Development by Light (W. R. Briggs, R. L. Health, and E. M. Tobin, Eds.), pp. 134–143. Am. Soc. Plant Physiol. Lauter, F-R., and Russo, V. E. A. 1990. Light-induced dephosphorylation of a 33kDa protein in the wild-type strain of Neurospora crassa: The regulatory mutants wc-1 and wc-2 are abnormal. J. Photochem. Photobiol. B: Biol. 5: 95–103. Lauter, F-R., Yarden, O., Yamashiro, C., Marchfelder, U., Russo, V. E. A., and Yanofsky, C. 1996. Dual photoregulation of cot-1, a kinaseencoding gene involved in hyphal growth in Neurospora crassa. [Abstract No. IV/o3. Abstract of Book of International Meeting on UV/Blue Light: Perception and Responses in Plants and Microorganisms, Philipps-Universita¨t, Marburg, Germany]. Liscum, E., and Briggs, W. R. 1995. Mutations in the NPH1 locus of Arabidopsis disrupt the perception of phototropic stimuli. Plant Cell 7: 473–485. Lo`pez-Diaz, I., and Cerda`-Olmedo, E. 1980. Relationship of photocarotenogenesis to other behavioural and regulatory responses in Phycomyces. Planta 150: 134–139. Ma¨tschke, M., Riedel, J., and Tischner, R. 1997. Evidence for signal transduction in the stimulation of nitrate uptake by blue light in Chlorella saccharophila. Photochem. Photobiol. 66: 128–132. Mehta, B. J., Salgado, L. M., Bejarano, E. R., and Cerda´-Olmedo, E. 1997. New mutants of Phycomyces blakesleeanus for -carotene production. Appl. Environ. Microbiol. 63: 3657–3661. Mustilli, A. C., and Bowler, C. 1997. Tuning in to the signals controlling photoregulated gene expression in plants. EMBO J. 16: 5801–5806. Nishiwaki, S., Fujiki, H., Suganuma, M., Furuya-Suguri, H., Matsushima, R., Iida, Y., Ojika, M., Yamada, K., Uemura, D., Yasumoto, T., Schmitz, F. J., and Sugimura, T. 1990. Structure–activity relationship within a series of okadaic acid derivatives. Carcinogenesis 11: 1837–1841. Oda, K., and Hasunuma, K. 1994. Light signals are transduced to the phosphorylation of 15kDa proteins in Neurospora crassa. FEBS Lett. 345: 162–166. Oda, K., and Hasunuma, K. 1997. Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia. Mol. Gen. Genet. 256: 593–601. Pan, J-M., Haring, M. A., and Beck, C. F. 1996. Dissection of the blue-light-dependent signal-transduction pathway involved in gametic differentiation of Chlamydomonas reinhardtii. Plant Physiol. 112: 303–309. Pan, J-M., Haring, M. A., and Beck, C. F. 1997. Characterization of blue light signal transduction chains that control development and maintenance of sexual competence in Chlamydomonas reinhardtii. Plant Physiol. 115: 1241–1249. Prokisch, H., Yarden, O., Dieminger, M., Tropschug, M., and Barthelmess, I. B. 1997. Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2⫹ gradient. Mol. Gen. Genet. 256: 104–114.
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Raugei, G., Dohrmann, U., Pohl, U., and Russo, V. E. A. 1982. Kinetics of photoaccumulation of -carotene in Phycomyces blakesleeanus. Planta 155: 296–300. Reymond, P., Short, T. W., Briggs, W. R., and Poff, K. L. 1992. Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 89: 4718–4721. Ruiz-Hidalgo, M. J., Benito, E. P., Sandmann, G., and Eslava, A. P. 1997. The phytoene dehydrogenase gene of Phycomyces: Regulation of its expression by blue light and vitamin A. Mol. Gen. Genet. 253: 734– 744. Salomon, M., Zacherl, M., and Ru¨diger, W. 1997. Assymetric, blue light-dependent phosphorylation of a 116-kilodalton plasma membrane protein can be correlated with the first- and second-positive phototropic curvature of oat coleoptiles. Plant Physiol. 115: 485–491. Scho¨nthal, A. 1992. Okadaic acid—A valuable new tool for the study of signal transduction and cell cycle regulation? New Biol. 4: 16–21.
213 Scott, K., and Zuker, C. 1997. Lights out: Deactivation of the phototransduction cascade. Trends Biochem. Sci. 22: 350–354. Sheen, J. 1993. Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO J. 12: 3497–3505. Short, T. W., and Briggs, W. R. 1994. The transduction of blue light signals in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 143–171. Toda, T., Shimanuki, M., and Yanagida, M. 1993. Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control. EMBO J. 12: 1987–1995. Yarden, O., Plamann, M., Ebbole, D. J., and Yanofsky, C. 1992. cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J. 11: 2159–2166. Yoshida, S., Ikeda, E., Uno, I., and Mitsuzawa, H. 1992. Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. Mol. Gen. Genet. 231: 337–344.
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Blue Light Signaling Chains in Phycomyces: Phototransduction of Carotenogenesis and Morphogenesis Involves Distinct Protein Kinase/Phosphatase Elements
George Tsolakis,* Eleni Parashi,* Paul Galland,† and Kiriakos Kotzabasis*,1 *Department of Biology, University of Crete, P.O. Box 2208, 71409 Heraklion, Crete, Greece; and †Fachbereich Biologie, Philipps-Universita¨t Marburg, Lahnberge, 35032 Marburg, Germany
Accepted for publication November 9, 1999
The reversible, dynamic phosphorylation of Ser, Thr, Tyr, or His residues of key proteins is a widely utilized mechanism for regulating a plethora of cellular events. Processes that are tightly controlled by protein phosphorylation require the catalytic activity of specific protein kinase(s) (PrK) for the addition of phosphate and specific protein phosphatase(s) (PrP) for the removal of phosphate. In principle, the steady-state level of phosphate in a target protein substrate can be modulated by the activities of the cognate PrK or PrP or both. The balance of these enzymatic activities defines in turn the strength and duration of the physiological event (Hunter, 1995). Protein phosphorylation as a regulatory mechanism can ensure signal relaying and amplification as well as signal(s) integration (e.g., the mitogen-activated protein kinase module; Ferrell, 1996); moreover, it can also offer efficient deactivation by switching the signal(s) off [e.g., the phototransduction cascade of vertebrates/invertebrates (Scott and Zucker, 1997)]. During the past decade, exciting studies implicating protein phosphorylation in blue light (BL)2-regulated phenomena of plants and fungi have been conducted. Among them, phototropism has gained the most attention. In several mono- and dicotyledonous plants, the BL-dependent protein phosphorylation of a plasma membrane protein of about 120 kDa in vivo and in vitro is well
Tsolakis, G., Parashi, E., Galland, P., and Kotzabasis, K. 1999. Blue light signaling chains in Phycomyces: phototransduction of carotenogenesis and morphogenesis involves distinct protein kinase/phosphatase elements. Fungal Genetics and Biology 28, 201–213. Carotenogenesis and morphogenesis represent two of the several responses sensitive to blue light which characterize the lower eukaryote Phycomyces blakesleeanus. Speculating that reversible phosphorylation may be an intracellular event beyond the photoperception step, we resorted to the use of first-choice inhibitors of protein phosphatases and protein kinases. The mycelial -carotene content of dark-grown cultures was induced by all agents administered, while the morphogenic output showed the typical trend effected by light only with one of the protein kinase inhibitors. Our data provide convincing evidence that protein phosphorylation plays a regulatory role in photocarotenogenesis and photomorphogenesis of Phycomyces. According to the model we propose, the putative signaling elements involved are anticipated to have a repressive function in the dark so that the responses are maintained in the ‘‘off ’’ mode until the moment photon information has to flow through the regulatory circuit. r 1999 Academic Press
1
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To whom correspondence should be addressed. Fax: 0030-81-394408. E-mail: [email protected].
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Abbreviations used: BL, blue light; OKA, okadaic acid; GEN, genistein; STS, staurosporine.
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established and represents an early tie in the transduction chain for phototropism (reviewed by Short and Briggs, 1994). Evidence in support of this connection first stemmed from in vitro studies with the nonphototropic hypocotyl (nph) mutants of Arabidopsis (Reymond et al., 1992; Liscum and Briggs, 1995). The determination of an asymmetrical microdistribution of the BL-induced protein phosphorylation at the irradiated versus the shaded side of Avena coleoptiles (Salomon et al., 1997) also strongly correlated the signaling aspect of phototropism with the dynamics of protein phosphorylation. Recently, the 120kDa phosphoprotein was suggested to be encoded by the nph1 gene of Arabidopsis, whose predicted amino acid sequence in the carboxy terminus contains motifs typical of a Ser–Thr PrK family within the PrK C group (Huala et al., 1997). In addition, an involvement of PrPs has been demonstrated for light-regulated gene expression in plants. A stimulatory role of PrP1 and PrP2A appears essential in the light-dependent expression of photosynthesis genes of maize (Sheen, 1993). Similarly, in barley, PrP1 and/or PrP2A is required to activate the BL-responsive promoter of the chloroplast psbD gene and the expression of other light-sensitive plastid- or nuclear-encoded genes (Christopher et al., 1997). For the ascomycete Neurospora, roles for PrK and PrP in BL transduction have been reported. Biochemical analyses demonstrating that light affects the phosphorylation states of several mycelial proteins with patterns in the wild type distinct from patterns of the BL-insensitive white collar mutants are notable (Lauter and Russo, 1990; Oda and Hasunuma, 1994). Furthermore, the recently described psp mutant, which completely lacks the ability to phosphorylate a 15-kDa protein as a normal response to BL, has offered a biochemical model system for further molecular analysis (Oda and Hasunuma, 1997). Further evidence of a close link between BL-induced phosphorylation and BL-sensitive physiological processes has been established for microalgae (Ma¨tschke et al., 1997; Pan et al., 1997). Most of the aforementioned studies prompted us to investigate the possible role of protein phosphorylation in two BL-sensitive physiological processes of Phycomyces blakesleeanus, carotenogenesis and morphogenesis. The photosignaling aspect of both responses is well described genetically (reviewed in Corrochano and Cerda-Olmedo, 1992) but the biochemical or molecular nature of the involved components remains unknown.
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Tsolakis et al.
We combined the ease in experimental manipulations Phycomyces offers with a pharmacological approach, namely, systematic feeding with widely used cell-permeable inhibitors of the enzymes that catalyze changes in phosphorylation. Our findings provide initial evidence that protein phosphorylation events are part of the network of activities brought into action by BL in this organism.
MATERIALS AND METHODS Chemicals. Okadaic acid (OKA) and okadaol were obtained from Calbiochem-Novabiochem Corp. (La Jolla, CA); genistein (GEN) and daidzein were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, PA); NaF and staurosporine (STS) were from Sigma Chemical Co. (St. Louis, MO). All other chemicals were obtained from Sigma and Merck (Frankfurt, Germany). Organism and culture conditions. All experiments were performed with the standard wild-type strain NRRL1555(-) of P. blakesleeanus on minimal agar plates (8 cm in diameter; Cerda-Olmedo, 1987). For convenience in handling, the solidified content (25 ml) of each dish was covered with circularly cut dialysis membrane (Visking C/110; Serva, Heidelberg, Germany) of the same diameter, which was autoclaved and then rinsed in water/liquid minimal medium. Each plate received 105 heat-activated spores (48°C, 10 min) in water suspension, which were carefully spread onto the membrane disk, according to Corrochano and Cerda-Olmedo (1988). Inoculated dishes were incubated without their lids in foil-wrapped cardboard boxes (four plates per box; the volume of the box with its cover was 14 L). All boxes were kept in a dark, air-conditioned place at 22 ⫾ 1°C. Treatments. Stock solutions of the various inhibitors were prepared in DMSO solution (2.5% v/v for okadaic acid and okadaol or 50% v/v for genistein, daidzein, and staurosporine) and stored as aliquots at ⫺20°C. Sodium fluoride stock solution was prepared in sterile 0.02% v/v Tween 80 water solution and maintained at room temperature. To prepare treatment solutions, all stock aliquots of the appropriate volume were further diluted immediately before use in sterile 0.02% v/v Tween 80 water solution (pH adjusted to 7.0 with a few drops of a 10 N solution of NaOH). Care was taken that the DMSO concentration in the final treatment solution was always adjusted to 0.05% v/v for okadaic acid and okadaol or 1% v/v for the other
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inhibitors in all doses administered and in control treatments (no inhibitor). The administration of a treatment solution took place 43 h after inoculation in a light-tight room under dim red-safe light obtained through a red filter (Plexiglass GS Rot 501, 3 mm thick; Ro¨hm GmbH, Darmstadt, Germany). A 1-ml aliquot of a treatment solution was added to each plate by injection between the membrane disk and the surface of the solid medium. In the light experiments, each cardboard box was uncovered, screened with a blue filter sheet (Plexiglass GS Blau 610, 3 mm thick; Ro¨hm GmbH), and given a 2-min light pulse 5 h after the injection (48th h of the culture) in the same light-tight room. The light source above the blue filter was a 15-W cool-white fluorescent lamp (TL-D, Super 80; Philips, Holland) and the BL energy flux reaching the plates was adjusted in situ to 0.02 J m⫺2 by the use of a photoradiometer system (IL760/IL1700/IL780; International Light, Newburyport, MA). In the dark experiments, culture plates were treated exactly as the BLirradiated plates, except that the light source was not turned on. Measurements of -carotene. The extraction protocol applied in mycelia 72 h after inoculation was essentially as described by (Raugei et al., 1982) with the following modifications: The preweighted mycelial portion of a plate intended for extraction was immersed in 9 ml ice-cold methanol, cut into smaller pieces instead of being powdered in a mortar with liquid nitrogen, and extracted as such. The other portion of the mycelial pad was used for dry weight determination after baking at 110°C for 14 h. All methanolic extractions were performed at 65°C. Morphogenesis assays. Morphogenic assays were carried out as described elsewhere (Corrochano and CerdaOlmedo, 1988). All macrophores from each plate were collected with forceps at the 96th h of cultures and their dry weights measured after drying at 110°C for 14 h. The visible sporangia of microphores were counted on the same macrophore-free plates under a 40⫻ stereomicroscope through calibrated grids set up within the eyepieces. Mean microphore density per square millimeter was calculated from counts of six to eight randomly chosen areas (24–32 quadrats sampling) on the mycelial surface of a plate. Statistical analyses. Treatments were repeated two to four times with two or four plates per experiment. Data from independent experiments were analyzed by the one-way analysis of variance (ANOVA) procedure at ␣ ⫽ 0.05. In cases in which the ANOVA results suggested at least one equality among the means of the treatment
effects, the Fisher multiple comparison procedure (LSD test) was applied among these treatment groups at the ␣ ⫽ 0.05 or 0.01 levels. Results obtained by the Fisher test are given in the figure legends. All analyses were performed with the SYSTAT software (version 5.0; Systat Inc., 1992).
RESULTS AND DISCUSSION Signal transduction studies usually exploit the biochemistry of signaling. Thus, a signaling chain can be made to work or not work by challenging the system under study, in the presence/absence of the primary stimulus, with agents (biochemical analogues, inhibitors, activators) known to influence the putative activity of interest involved in the information flow. A representative breakthrough that ensued from this approach was the identification of signaling intermediates in phytochrome pathways (reviewed by Mustilli and Bowler, 1997). We decided to apply the same experimental rationale to P. blakesleeanus, a lower eukaryote long known mainly for the complex behavior of its sporangiophores (fruiting bodies) in response to blue light (reviewed in Galland, 1990). Two easily assayable photoresponses of this organism were chosen as markers in our experiments: morphogenesis and carotenogenesis. Morphogenesis is a developmental process that can be characterized by the dry weight of macrophores (giant sporangiophores) and the number of microphores (dwarf sporangiophores). Carotenogenesis refers to the mycelial accumulation of -carotene. Illumination, especially BL, increases the macrophore yield and the -carotene content but decreases the production of microphores. The biochemical and molecular properties of the BL signaling network operating in these responses remain elusive. Speculating that reversible phosphorylation may be an intracellular event beyond the primary activation of BL receptors, we present here data from experiments in which inhibitors of PrP and PrK were used. In all treatments reported below, the culture conditions employed were those recommended by Corrochano and Cerda-Olmedo (1988). A main advantage of this surface culture system is that both photomorphogenesis and photocarotenogenesis exhibit simultaneous periods of maximum photocompetence (Bejarano et al., 1990). Therefore, using the same batch of growing culture plates, all responses can be recorded at the proper developmental moment without special requirements for each separate response.
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The Effect of PrP Inhibitors on Dark-Grown Cultures Millimolar concentrations of the widely used PrP inhibitor NaF significantly stimulated the -carotene content of dark-grown mycelia in a dose-dependent manner (Fig. 1). The maximal stimulation was seen between 1 and 40 mM NaF, despite the apparent plateau (⬃60% above dark control values) from 1 to 14 mM and the declining trend above 4 mM. Up to 1 mM NaF, both morphogenic variables were insensitive to it, but above 4 mM, a developmental behavior typical of growth under unfavorable conditions was observed (Corrochano and CerdaOlmedo, 1988): increase in the number of microphores and reduced macrophorogenesis. This behavior should be attributed to fluoride toxicity and should not be considered response-specific since, in the same concentration range, -carotene levels are also declining. According to the general rationale given above, mimicking light action under dark conditions with a chemical of known action against an intracellular target constitutes a
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gross but not sufficient hint that the same type of action on this target is also effected by photons. Thus, BL information in mycelia must be transduced at least via a kind of PrP inhibition for carotenogenesis. This PrP involvement may be an event irrelevant to the chains determining the morphogenic decisions, since the morphogenic output appeared unaltered after the fluoride treatment in the concentration range in which (skoto)carotenogenesis was manifested. The positive effect of fluoride on mycelial carotenogenesis of Phycomyces was first reported by Desai et al. (1973). They correlated the fluoride stimulation in carotene level with a considerable drop in the total activities of acid phosphatase and pyrophosphatase. These phosphatases were thus suggested to be part of the mechanism controlling the biosynthetic flux to -carotene. In a more recent study of alkaline phosphatase from Phycomyces, it was also noted that tetramisole, a potent inhibitor of this enzyme, could increase -carotene of macrophores up to fivefold when included in solid minimal medium (cited in Cohen, 1985).
FIG. 1. Effects of NaF on mycelial -carotene, macrophores dry mass, and microphores numbers of dark-grown cultures. Points and bars (mean ⫾ SE) with common letters at their side define groups of statistical similarity among the corresponding treatments at ␣ ⫽ 0.05 by Fisher’s multiple comparison procedure. The absolute mean values of dark controls that were set as 100% for each variable were -carotene, 72.17 ⫾ 3.15 µg/g of dry mycelial weight; macrophores dry weight, 125 ⫾ 1.108 mg/plate; microphores density, 56 ⫾ 3 mm⫺2.
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Blue Light Signaling in Mycelia of Phycomyces
All the above led us to experiment with OKA, a reagent that does not inhibit acid or alkaline phosphatases (Hardie, 1993). This 38-carbon fatty acid acts as a noncompetitive or mixed inhibitor of serine/threonine PrP1 and PrP2A and is useful because the PrP1/PrP2A sensitivities to OKA have been substantially conserved in most eukaryotic cells (Cohen et al., 1990). The dose–response study of Fig. 2 supports the fluoride data. Up to 0.5 µM OKA, -carotene levels rise over dark control levels up to ⬃70%, while the vein of increase reappeared above a small plateau that extends between 0.5 and 1 µM OKA doses. Both morphogenic variables were insensitive to all okadaic acid concentrations provided, as was the case for NaF. Okadaol, an okadaic acid analogue with approximately 40% of the inhibitory activity of okadaic acid in vitro (Nishiwaki et al., 1990), was effective at 1 µM, increasing -carotene by ⬃25% above dark control values (data not shown). The in vitro IC50 values of PrP2A and PrP1 for OKA
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differ by 3 to 100 times, depending on the cell type, with IC50 values of PrP2A being lower than those of PrP1. Because of this difference, low okadaic acid concentrations are usually expected to inhibit PrP2A selectively in vivo, while a PrP1 inhibition is expected at higher okadaic acid concentrations (Scho¨nthal, 1992). In this respect, a PrP2A/ PrP1 involvement in BL-mediated signaling of carotenogenesis in Phycomyces cannot be ruled out, granted that the participation of these PrPs in cellular regulation is widespread. The present okadaic acid dose–response analyses would be more informative if IC50 values from a Phycomyces cell-free system were available. For this reason, the assignment of selective PrP function to photocarotenogenesis is premature. The notion, however, that two different regulatory activities might be brought into action in a negative way (by their light-mediated inhibition) fits well with the property of photocarotenogenesis being a twostep response with two separate components (Bejarano et al., 1990).
FIG. 2. The inducing effect of OKA on carotenogenesis under dark-growth conditions. Averages accompanied by a letter are significantly different from dark control -carotene levels at the ␣ ⫽ 0.05 level by Fisher’s test. Both morphogenic variables were insensitive to OKA treatment, with ANOVA P values of 0.686 for macrophores and 0.737 for microphores. At all doses plus controls, DMSO was in 0.05% v/v final concentration. Absolute dark control averages: -carotene, 67.053 ⫾ 3.67 µg/g of dry mycelial weight; macrophores dry weight, 116 ⫾ 3.45 mg/dish; microphores counts, 62 ⫾ 2 mm⫺2.
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Analyses with PrK Inhibitors in the Dark The net effect produced intracellularly by an OKA application is a state of increased phosphorylation via the primary inhibition of PrPs and the minor action of the opposing PrKs, provided that they are partially active. Shifting the balance of reversible protein phosphorylation toward a hypophosphorylation state, however, would allow us to see the other side of the picture. To this effect, dark-grown cultures were incubated with PrK inhibitors. STS was first choice because it is a ‘‘catch-all’’ compound inhibiting both serine/threonine- and tyrosine-specific PrKs (Hidaka and Kobayashi, 1993). A dose–response analysis with doses covering three orders of magnitude showed that all three variables under study were susceptible to STS (Fig. 3). At 10 µM, STS was able to induce -carotene synthesis by 50% or increase macrophores dry weight by ⬃35% above the corresponding dark control values. A parallel maximum decrease ⬃60% below dark control
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values was also observed for microphorogenesis at 10 µM STS. The simultaneous STS-evoked variations of microphorogenesis and carotenogenesis, according to the pattern elicited by light, imply that the STS-inhibited target(s) may be, likewise, light-inhibited and a common element in the photocascades operating for these responses. That BLactivated transduction chains in Phycomyces share or are based functionally on common gene products is well established by studies with mutants (Corrochano and Cerda-Olmedo, 1992). Moreover, evidence that heterotrimeric G proteins constitute a common tie in BL signaling circuits regulating morphogenesis, carotenogenesis, and gene expression is strong (G. Tsolakis and K. Kotzabasis, unpublished data). Identification of the intracellular PrK activity(-ies) inhibited by STS is not possible with our in vivo data. In yeasts, however, STS concentrations from 2 to 70 µM have been reported to be sufficient to target
FIG. 3. The effect of staurosporine on carotenogenesis and morphogenesis of dark-grown cultures. Any two averages within each curve that do not have at least one same letter are significantly different at ␣ ⫽ 0.05 by the LSD test. Pairwise comparisons of all points on the macrophores curve showed significant differences at ␣ ⫽ 0.01 level. In all doses plus controls, the final DMSO concentration was 1% v/v. Absolute dark control values were: -carotene, 73.47 ⫾ 4.9 µg/g of dry mycelial weight; macrophores dry mass, 125.5 ⫾ 1.07 mg/dish; microphores counts, 54 ⫾ 2 mm⫺2.
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Blue Light Signaling in Mycelia of Phycomyces
intracellularly the regulatory activity of PrK C, thus implicating its participation in various cell functions (Yoshida et al., 1992; Toda et al., 1993; Branda˜o et al., 1994; Kobori et al., 1994). The only cellular process known to date in Phycomyces that implicates PrK C is spore germination. The role of this signaling regulator was probed by a PrK C-specific activator (a phorbol ester) which prevented the germination process in an early phase after spore activation either by acetate or by heat shock (Carillo-Rayas et al., 1988). Although the authors did not discuss the possibility that a PrK C activity may have to be repressed for the germination to proceed normally after the activating signal, an analogy with the present study—i.e., the use of a PrK inhibitor as a response elicitor—is easily drawn. Notable also is another study wherein STS mimicked in dark-grown cultures of Chlamydomonas the BL-dependent gamete maturation (Pan et al., 1996). In this photosignaling process, an active PrK C-like component was inferred to restore the conversion of pregametes to gametes blocked in the dark. In addition, illumination was suggested to overcome this blockade—probably through an inhibiting effect on the blocking activity—since PrK C-specific activators indeed inhibited gametogenesis in the light. To further evaluate the behavior of STS, genistein, another PrK inhibitor effective against tyrosine-specific PrKs, was utilized. Dark-grown plates given GEN concentrations ranging from 1 to 100 µM showed an increasing trend for mycelial -carotene; maximum levels ⬃70% above the dark control values were obtained at 100 µM GEN (Fig. 4A). The morphogenic expression remained neutral along this treatment range (Fig. 4B). Parallel experiments with daidzein, a negative control compound for genistein (Akiyama et al., 1987), confirmed the specificity of GEN on carotenogenesis and its inability to affect both morphogenic variables (Figs. 4A and 4B). The inducing effect of GEN on carotenogenesis suggests that the light signaling circuit of this response could involve a protein tyrosine kinase additional to those disclosed via OKA or STS. That both PrK/PrP inhibitors are able to promote -carotene levels in the dark indicates indirectly the complexity of the transduction process and, in part, differentiates photomorphogenesis from photocarotenogenesis in the requirement for these putative component(s).
Combinatorial Experiments: An Approach to Order the Effective Components The stimulating effect of the various inhibitors in the dark, described above, suggests that their corresponding
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intracellular targets exist and normally operate under dark conditions, so that mycelial responses are kept ‘‘switched off.’’ These putative signaling components must provide through their catalytic action an overall phosphorylation state for mycelia that is informative of the ambient dark. It seemed reasonable that this default dark-created phosphorylation state might respond either to BL or to the use of PrP/PrK inhibitors. Thus, to test this hypothesis, we included the primary stimulus in the inhibitor treatments (Table 1). An exposure to a nonsaturating BL flux— according to the analyses by Corrochano and CerdaOlmedo (1990)—was chosen with a view to let the responses be expressed quantitatively, avoiding any masking effects caused by light alone. The inhibitor doses tried were those that produced near maximal (okadaic acid, okadaol) or maximal (the other agents) dark output, as determined above. Okadaic acid or genistein in combination with BL (treatments iii and vi of Table 1) turned out to be the most effective treatments in the induction of -carotene (40– 55% above the light control, treatment i of Table 1). The -carotene output from the rest of inhibitors plus BL (treatments ii, iv, and v in Table 1) was weaker, about half of that produced by GEN or OKA plus BL, but also differed significantly from that of the light control (20–26% above light control levels). Concerning photomorphogenesis, only staurosporine (treatment v in Table 1) of all the inhibitors appeared potent enough to increase macrophores significantly (⬃20%) above light control levels or to reduce microphore density by ⬃60% below the corresponding light control output. The synergism between BL and the PrP/PrK inhibitors used discloses that the short light action could be enhanced by a sustained state effected intracellularly via these inhibitors. This enhancement (as measured by the efficiency of the responses) confirms that the intracellular targets of these compounds occupy intermediary positions within the cascades photoregulating the responses under study. Moreover, the observed interaction between BL and inhibitors constitutes another argument in favor of the prediction that photon energy acts negatively on the default PrP/PrK activities to ‘‘switch on’’ the responses. The special sensitivity of morphogenesis to staurosporine as opposed to the indistinct induction of carotenogenesis by all inhibitors, either in the dark or in the light, suggested the experiments of Table 2. Feeding the system with the various inhibitors in combination facilitates the
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FIG. 4. The specific positive effect of genistein (A) on carotenogenesis in dark-grown cultures. This inhibitor treatment had no significant output at the ␣ ⫽ 0.01 level (B) from either macrophorogenesis (ANOVA P ⫽ 0.862) or microphorogenesis (ANOVA P ⫽ 0.431). All variables were unaffected by daidzein (A and B), with nonsignificant differences from dark controls (ANOVA P values: -carotene, 0.991; macrophores, 0.906; microphores, 0.999). Final DMSO concentration and absolute dark control averages as in Fig. 3.
identification of the biochemical entities involved and the development of a hypothetical function scheme. Combining GEN or OKA with STS (treatments vi or vii of Table 2) produced in the dark as much mycelial -carotene as that produced by each of the three inhibitors separately (treatments ii, iii, and iv in Table 2). In contrast,
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the combination of GEN and OKA was particularly productive in -carotene and significantly differed from any other combinatorial or single treatment. Hence, in carotenogenesis, the absence of synergism between STS and GEN or OKA is worthy of note, given that -carotene is induced by all three inhibitors separately. As a possible explanation of
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TABLE 1 Inhibitor Treatments Combined with a BL Pulse Treatment typea i. ii. iii. iv. v. vi. vii.
BL Fluoride (1 mM) ⫹ BL Okadaic acid (1 µM) ⫹ BL Okadaol (1 µM) ⫹ BL Staurosporine (10 µM) ⫹ BL Genistein (100 µM) ⫹ BL Daidzein (100 µM) ⫹ BL
-Carotene
Macrophores
Microphores
100.00b ⫾ 3.870 A 126.75 ⫾ 10.350 B 137.58 ⫾ 6.181 B,C 121.48 ⫾ 6.260 B 124.81 ⫾ 8.100 B 155.30 ⫾ 7.260 C 111.57 ⫾ 3.440 A,B
100.0c ⫾ 1.750A 103.93 ⫾ 1.865 A 100.74 ⫾ 1.730 A 100.41 ⫾ 2.320 A 119.12 ⫾ 1.850 103.50 ⫾ 1.900 A 103.73 ⫾ 1.470 A
100.00d ⫾ 5.151 A 100.05 ⫾ 6.720 A 98.92 ⫾ 4.924 A 102.03 ⫾ 2.850 A 38.30 ⫾ 4.160 99.72 ⫾ 5.290 A 100.38 ⫾ 3.220 A
Note. Dark-grown dishes of Phycomyces blakesleeanus were provided at the 43rd h of culture with 1 ml of the listed reagents and 5 h later were illuminated with a BL flux of 0.02 J m⫺2 for 2 min. Mean ⫾ SE values are expressed in relation to BL control averages which were set at 100%. Significant differences (P ⬍ 0.05) by Fisher’s test are indicated by different capital letters for any two treatment mean values. Absolute dark control averages of all measured variables: 64.63 µg of -carotene/g dry mycelial wt; 0.114 g of dry macrophore mass/plate; 63 microphores/mm2. a Final DMSO concentration 1% v/v in all treatments. b 110.97 µg of -carotene/g dry mycelial wt. c 0.147 g of dry macrophore mass/plate. d 26 microphores/mm2.
the effects of the STS/GEN and STS/OKA treatments on levels of carotenogenesis, the action of one inhibitor seems to mask or block the action of the other. Our statistical analysis, however, cannot discriminate between the action that masks and the action that is masked, but some kind of interaction(s) is hidden. Despite the obscure nature of the interaction, the carotenogenic behavior of the STS/OKA and STS/GEN combinations may be justified. We reason that a masking action could be developed only if the signaling elements targeted simultaneously by STS and OKA or GEN act in the same pathway for controlling carotenogenesis and probably at different regulatory steps.
By analogy, the synergistic interaction between OKA and GEN in carotenogenesis could stem from the action of these agents on signaling components that are not colinear between them but either on the same pathway as the STS-sensitive target. The insensitivity of both morphogenic variables to GEN or OKA is once more confirmed by comparing the pairwise combinations with the STS treatment (treatments v, vi, and vii versus iv in Table 2). If the morphogenic output of the ‘‘light treatments’’ (Table 1) is also taken into account, then the STS-targeted element could be shared by the photosignaling cascade operating for morphogenesis.
TABLE 2 Combinatorial Inhibitor Treatments in Dark-Grown Cultures of Phycomyces blakesleeanus Treatment typea i. ii. iii. iv. v. vi. vii.
Dark control GEN (1 µM) OKA (0.1 µM) STS (5 µM) GEN (1 µM) ⫹ OKA (0.1 µM) GEN (1 µM) ⫹ STS (5 µM) OKA (0.1 µM) ⫹ STS (5 µM)
-Carotene
Macrophores
Microphores
100.00b ⫾ 5.042 150.85 ⫾ 10.400 A 140.22 ⫾ 12.930 A 137.60 ⫾ 5.600 A 177.70 ⫾ 6.348 152.51 ⫾ 8.610 A 148.90 ⫾ 3.960 A
100.00c ⫾ 3.131 A 97.80 ⫾ 2.731 A 100.26 ⫾ 4.800 A 126.00 ⫾ 0.518 B 99.83 ⫾ 0.579 A 125.00 ⫾ 1.150 B 124.00 ⫾ 0.326 B
100.00d ⫾ 3.105 A 99.50 ⫾ 2.700 A 106.95 ⫾ 8.200 A 44.98 ⫾ 0.341 B 97.29 ⫾ 1.280 A 45.43 ⫾ 0.747 B 45.65 ⫾ 0.587 B
Note. Mean ⫾ SE values are shown as percentages of the dark control averages. Statistical similarity grouping among the treatments within each variable according to LSD analyses (P ⬎ 0.05 for carotenogenesis, P ⬎ 0.01 for macro- and microphorogenesis) is defined by common capital letters. a Final DMSO concentration 1% v/v in all treatments. b 64.63 µg of -carotene/g dry mycelial wt. c 0.114 g of dry macrophore mass/plate. d 63 microphores/mm2.
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A Simple Phototransduction Scheme Our data can be most conveniently summarized by arranging the PrP/PrK elements on a branched phototransduction pathway which controls carotenogenesis and morphogenesis (Fig. 5). The so-called pic mutants characterized by a specific defect in the photoinduction of -carotene behave normally with respect to other photoresponses (Lopez-Diaz and Cerda-Olmedo, 1980). In addition, the pim mutants develop microphores even under continuous illumination, which normally inhibits them (Flores et al., 1998). Both these mutants corroborate the notion of a branched photosignaling pathway for carotenogenesis and morphogenesis. Thus, one needs to assume that the pic products act exclusively on the photocarotenogenic branch either before or after the requisite PrTyrK/PrP junction. By analogy, the pim gene products should be located functionally beyond the branchpoint of Fig. 5, exclusively on the morphogenic branch, which remained concealed from our experimental approach. The hierarchy shown in the proposed model, with the STS-sensitive component occurring before the OKA/GEN targets, is not arbitrary. Since the unknown PrK (octagon in
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Fig. 5) via its inhibition by STS potentiates expression of all the response markers for BL signaling, this STS-mediated inhibition should be an early event in the sensory pathway. How close this STS-targeted step lies to the initial event of BL receptor(s) activation(s) cannot be inferred. In the carotenogenic BL-relaying branch, however, by putting the OKA/GEN-sensitive steps downstream of the STSsensitive element, two requirements are met. First, a signaling dissimilarity between carotenogenesis and morphogenesis is obtained according to their differential reactions to the agents used, and second, the hint of the masking relationship discussed above is biased in favor of an OKA or a GEN action working as a mask over the STS action. The synergy between OKA and GEN actions is another theme depicted in the proposed model with two arrows starting from the OKA/GEN-inhibited targets and converging toward the -carotene output. The value of synergistic interactions, at least between signaling transduction pathways, in terms of lending responses greater sensitivity and rapidity has been discussed elsewhere (Fuglevand et al., 1996). Whether the proposed OKA/GENsensitive steps belong to independent photosignaling path-
FIG. 5. A simple model of the various inhibitor treatments and their interactions. The flux through the signaling circuit is given with arrows. The inhibiting action of the exogenous reagents on the proposed targets (polygons) is shown as T lines. T lines and question marks within arrows indicate the possible BL-effected, normal inhibition of the nearby activities. The protein phosphatase (PrP) and protein tyrosine kinase (PrTyrK) polygons are drawn downstream of the protein kinase (PrK) octagon to denote the differential sensitivity of carotenogenesis to OKA and GEN with relation to morphogenesis. The synergy GEN/OKA is indicated with two independent, final arrows converging on the -carotene output. The broken arrows within polygons reflect the proposed default, repressive action of the signaling components to keep the responses in the ‘‘off’’ mode in ambient darkness.
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Blue Light Signaling in Mycelia of Phycomyces
ways or to one branched pathway cannot be determined. Nonetheless, carotenogenesis is indeed characterized by greater photosensitivity relative to morphogenesis [their lowest thresholds are 10⫺5 and 10⫺4 J m⫺2, respectively (Bejarano et al., 1990; Corrochano and Cerda-Olmedo, 1990)] and is considerably more rapid [e.g., the phytoene dehydrogenase mRNA rises four- to sevenfold in the first 5 min of BL illumination (Ruiz-Hidalgo et al., 1997)]. The putative signaling elements included in Fig. 5 were all identified by inhibitors and this led to the reasoning that BL information may be transduced via these elements by inhibiting their activity. In other words, the action of these transduction components is to help keep the photobehavior ‘‘silent’’ in the dark. Such a type of negative regulation or repressive function in the absence of the light stimulus has been already proposed both for a PrK C-like component in the BL signaling chain operating for the gametic differentiation of Chlamydomonas (Pan et al., 1996) and for the Arabidopsis cop/det/fus mutants which develop light-grown morphologies in complete darkness (Kwok and Deng, 1996). Therefore, consisting not only of positive elements with a downstream activating role but also of negative elements (broken arrows within polygons in Fig. 5) whose light-signal blockade is lifted once the signal flows may be a general feature of aneural photosignaling cascades. The complexity and the interaction types reported here are not unexpected. The genetics of -carotene biosynthesis in Phycomyces has disclosed several gene loci which participate in the regulatory circuit through a variety of interactions, as suggested by numerous studies in mutant strains (Avalos et al., 1992, and references therein; Mehta et al., 1997). On the other hand, the established photosensory pathways of carotenogenesis and morphogenesis are also not lacking in complexity. (The current schemes of the photosensory pathways as defined by genetical data may be found at the following internet address: www.es.embnet.org/ ˜ genus/Phycotrans.html). A branched photocascade for these light responses has also been postulated on the basis of mutant analyses. The products of the genes madA and madB are required both for photocarotenogenesis and for photodifferentiation of sporangiophores (Jayaram et al., 1980; Lopez-Diaz and Cerda-Olmedo, 1980; Corrochano and Cerda-Olmedo, 1990). In the context of our model, these gene products could act either ‘‘upstream’’ or ‘‘downstream’’ of the STS-sensitive PrK. Their signaling position could not, however, be envisaged ‘‘downstream’’ of the proposed branchpoint, because this would mean their exclusive effect on the carotenogenic branch only. Our
model in Fig. 5 is also in accordance with the behavior of other photomutants of Phycomyces, which specifically show defective photocarotenogenesis or photomorphogenesis. That (photo)morphogenesis appeared to be sensitive only to STS does not imply that the intracellular target of this compound is also singular; it also does not imply that the unknown effector target downstream of the STSsensitive step is singular. Neurospora has so far shown that hyphal development normally requires two different PrKs (Yarden, 1992), with one exhibiting a peculiar photoexpression pattern (Lauter et al., 1996) and one PrP (Prokisch et al., 1997). Even if the Neurospora requirements have their counterparts in Phycomyces morphogenic processes, two strategies should be put in future practice. Apart from the development of the proper in vitro cell-free systems in Phycomyces that will identify photoregulated PrK/PrP activities, the use of more specific PrK/PrP substances both in the wild type and in the various mutants will allow us to map the disclosed elements as to the classical gene loci that define the photosensory pathways.
ACKNOWLEDGMENTS We thank Dr. Alexandros Argirokastritis for his useful suggestions and comments on the manuscript.
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212 Cerda´-Olmedo, E. 1987. Standard growth conditions and variations. In Phycomyces (E. Cerda´-Olmedo and E. D. Lipson, Eds.), pp. 337–339. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Christopher, D. A., Xinli, L., Kim, M., and Mullet, J. E. 1997. Involvement of protein kinase and extraplastidic serine/threonine protein phosphatases in signaling pathways regulating plastid transcription and the psbD blue light-responsive promoter in barley. Plant Physiol. 113: 1273–1282. Cohen, P., Holmes, C. F. B., and Tsukitani, Y. 1990. Okadaic acid: A new probe for the study of cellular regulation. Trends Biochem. Sci. 15: 98–102. Cohen, R. J. 1985. Alkaline phosphatase from Phycomyces blakesleeanus. Exp. Mycol. 9: 195–202. Corrochano, L. M., and Cerda´-Olmedo, E. 1988. Photomorphogenesis in Phycomyces: Dependence on environmental conditions. Planta 174: 309–314. Corrochano, L. M., and Cerda´-Olmedo, E. 1990. Photomorphogenesis in Phycomyces: Competence period and stimulus–response relationships. J. Photochem. Photobiol. B:Biol. 5: 255–266. Corrochano, L. M., and Cerda´-Olmedo, E. 1990. Photomorphogenesis in behavioural and colour mutants of Phycomyces. J. Photochem. Photobiol. B: Biol. 6: 325–335. Corrochano, L. M., and Cerda´-Olmedo, E. 1992. Sex, light and carotenes: The development of Phycomyces. Trends Genet. 8: 268–274. Desai, H. G., Desai, J. D., and Modi, V. V. 1973. Regulation of carotenogenesis in Phycomyces blakesleeanus. Indian J. Microbiol. 13: 57–62. Ferrell, J. E. 1996. Tripping the switch fantastic: How a protein kinase cascade can convert graded inputs into switch-like outputs. Trends Biochem. Sci. 21: 460–466. Flores, R., Cerda´-Olmedo, E., and Corrochano, L. M. 1998. Separate sensory pathways for photomorphogenesis in Phycomyces. Photochem. Photobiol. 67: 467–472. Fuglevand, G., Jackson, J. A., and Jenkins, G. I. 1996. UV-B, UV-A, and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell 8: 2347–2357. Galland, P. 1990. Phototropism of the Phycomyces sporangiophore: A comparison with higher plants. Photochem. Photobiol. 52: 233–248. Hardie, D. G. 1993. Use of protein phosphatase inhibitors in intact cells. In Protein Phosphorylation: A Practical Approach (D. G. Hardie, Ed.), pp. 109–119. IRL Press, Oxford. Hidaka, H., and Kobayashi, R. 1993. Use of protein (serine/threonine) kinase activators and inhibitors to study protein phosphorylation in intact cells. In Protein Phosphorylation: A Practical Approach (D. G. Hardie, Ed.), pp. 87–107. IRL Press, Oxford. Huala, E., Oeller, P. W., Liscum, E., Han, I-S., Larsen, E., and Briggs, W. R. 1997. Arabidopsis NPH1: A protein kinase with a putative redox-sensing domain. Science 278: 2120–2123. Hunter, T. 1995. Protein kinases and phosphatases: The yin and yang of protein phosphorylation and signaling. Cell 80: 225–236. Jayaram, M., Leutwiler, L., and Delbru¨ck, M. 1980. Light induced carotene synthesis in mutants of Phycomyces with abnormal phototropism. Photochem. Photobiol. 32: 241–245. Kobori, H., Toda, T., Yaguchi, H., Toya, M., Yanagida, M., and Osumi, M.
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