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Phytochrome-regulated repression of gene expression requires calcium and cGMP

1997; Springer Nature; Volume: 16; Issue: 10 Linguagem: Inglês

10.1093/emboj/16.10.2554

1460-2075

Gunther Neuhaus, Chris Bowler, Kazuyuki Hiratsuka, Hiroshi Yamagata, Nam‐Hai Chua,

Plant tissue culture and regeneration

Article15 May 1997free access Phytochrome-regulated repression of gene expression requires calcium and cGMP Gunther Neuhaus Gunther Neuhaus Institut für Pflanzenwissenschaften, ETH-Zurich, Universitätstrasse 2, CH-8092 Zürich, Switzerland Institute for Biology II, Department of Cell Biology, Schaenzlestrasse 1, Freiburg, Germany Search for more papers by this author Chris Bowler Corresponding Author Chris Bowler Stazione Zoologica, Villa Comunale, I-80121 Napoli, Italy Search for more papers by this author Kazuyuki Hiratsuka Kazuyuki Hiratsuka Department of Molecular Biology, Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-01 Japan Search for more papers by this author Hiroshi Yamagata Hiroshi Yamagata Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada, Kobe, 657 Japan Search for more papers by this author Nam-Hai Chua Nam-Hai Chua Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA Search for more papers by this author Gunther Neuhaus Gunther Neuhaus Institut für Pflanzenwissenschaften, ETH-Zurich, Universitätstrasse 2, CH-8092 Zürich, Switzerland Institute for Biology II, Department of Cell Biology, Schaenzlestrasse 1, Freiburg, Germany Search for more papers by this author Chris Bowler Corresponding Author Chris Bowler Stazione Zoologica, Villa Comunale, I-80121 Napoli, Italy Search for more papers by this author Kazuyuki Hiratsuka Kazuyuki Hiratsuka Department of Molecular Biology, Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-01 Japan Search for more papers by this author Hiroshi Yamagata Hiroshi Yamagata Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada, Kobe, 657 Japan Search for more papers by this author Nam-Hai Chua Nam-Hai Chua Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA Search for more papers by this author Author Information Gunther Neuhaus2,3, Chris Bowler 4, Kazuyuki Hiratsuka5, Hiroshi Yamagata6 and Nam-Hai Chua1 1Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY, 10021-6399 USA 2Institut für Pflanzenwissenschaften, ETH-Zurich, Universitätstrasse 2, CH-8092 Zürich, Switzerland 3Institute for Biology II, Department of Cell Biology, Schaenzlestrasse 1, Freiburg, Germany 4Stazione Zoologica, Villa Comunale, I-80121 Napoli, Italy 5Department of Molecular Biology, Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara, 630-01 Japan 6Laboratory of Biochemistry, Faculty of Agriculture, Kobe University, Nada, Kobe, 657 Japan The EMBO Journal (1997)16:2554-2564https://doi.org/10.1093/emboj/16.10.2554 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The plant photoreceptor phytochrome A utilizes three signal transduction pathways, dependent upon calcium and/or cGMP, to activate genes in the light. In this report, we have studied the phytochrome A regulation of a gene that is down-regulated by light, asparagine synthetase (AS1). We show that AS1 is expressed in the dark and repressed in the light. Repression of AS1 in the light is likely controlled by the same calcium/cGMP-dependent pathway that is used to activate other light responses. The use of the same signal transduction pathway for both activating and repressing different responses provides an interesting mechanism for phytochrome action. Using complementary loss- and gain-of-function experiments we have identified a 17 bp cis-element within the AS1 promoter that is both necessary and sufficient for this regulation. This sequence is likely to be the target for a highly conserved phytochrome-generated repressor whose activity is regulated by both calcium and cGMP. Introduction Light is perceived in plants by three major classes of photoreceptors: the phytochromes, the blue/UVA receptors (cryptochromes) and the UVB receptors (Quail et al., 1995). Of these, the most intensively studied are the phytochromes, which exist in two photo-reversible forms: the red light absorbing form, Pr, generally considered to be physiologically inactive, and the far-red absorbing form, Pfr, known to mediate a broad range of plant responses to light (Quail et al., 1995; Smith, 1995; von Arnim and Deng, 1996). Some responses mediated by Pfr can be reversed by far-red light, which converts Pfr back to Pr. In higher plants, the phytochromes are encoded by multigene families (Quail et al., 1995). Each phytochrome is thought to have a different physiological role and the recent availability of mutants deficient in individual phytochromes is allowing further definition of these specificities (reviewed in Millar et al., 1994; Quail et al., 1995; von Arnim and Deng, 1996). Some responses have now been linked to particular phytochromes, although there nonetheless appears to be some overlap between the functions of individual phytochromes within any given plant species (Reed et al., 1994). The different phytochromes make up two distinct classes, known as type I and type II (Quail et al., 1995; Smith, 1995). Type I phytochromes are the most abundant in dark-grown plants, but they are light labile due to the rapid degradation and/or sequestration of the Pfr form in the light. In contrast, the type II phytochromes are present in much lower amounts, but their stability in the Pfr form ensures that they are predominant in light-grown plants. Hence, type I phytochrome is thought to play a specific role during the initial de-etiolation process, whereas type II may be more important for mediating phytochrome responses in mature plants. Phytochrome A (PHYA) is the only type I phytochrome to have been identified and it may in fact be the only molecular species within the type I pool (see Clack et al., 1994). Like the PHYA apoprotein, PHYA mRNA abundance also decreases in the light (see Sharrock and Quail, 1989, and references therein), particularly in monocotyledons, where down-regulation of PHYA gene expression has been found to be mediated by an autoregulatory mechanism involving phytochrome itself (Lissemore and Quail, 1988). In addition to PHYA, several other genes have been found to be down-regulated by light. These include genes encoding NADPH protochlorophyllide oxidoreductase (Mösinger et al., 1985), β-tubulin (Colbert et al., 1990; Tonoike et al., 1994; Leu et al., 1995), asparagine synthetase (AS1) (Tsai and Coruzzi, 1990, 1991), the homeodomain proteins Athb-2 and Athb-4 (Carabelli et al., 1993) and two genes denoted NPR1 and NPR2 in Lemna (Okubara et al., 1993). Phytochrome regulates these responses and two formal possibilities can be considered to account for how it does so (Bruce et al., 1991): (i) Pfr generates a repressor in the light; (ii) Pr generates an activator in the dark. Current knowledge of phytochrome function would tend to favour Pfr repression as the most likely mechanism, because much evidence implicates Pfr, and not Pr, in controlling many other responses. However, it has proved extremely difficult to design physiological experiments that could definitively distinguish between the two possibilities. In this report, we present the results of experiments that can discriminate between Pfr repression and Pr activation as possible mechanisms controlling the down-regulation of gene expression in the light. Specifically, we have studied the signal transduction events stimulated by PHYA to regulate expression of one of these negatively light regulated genes, AS1, using microinjection to deliver individual molecules into the cells of wild-type and aurea mutant tomato seedlings, as previously described (Neuhaus et al., 1993). PHYA is present in etiolated seedlings of the aurea mutant at 20% wild-type levels and is spectrally inactive, whereas PHYB (a type II phytochrome) is present and active at normal levels (Sharma et al., 1993). In contrast to the behaviour of wild-type seedlings, chloroplasts and anthocyanin pigments fail to develop within the hypocotyl cells of etiolated aurea seedlings in response to light. However, a wild-type phenotype can be restored to aurea hypocotyl cells by injection of exogenous PHYA (Neuhaus et al., 1993). This system therefore allows the manipulation and subsequent dissection of the signal transduction pathways used by PHYA by identifying agonists or antagonists of these responses. In this way, we have previously reported that the Pfr form of PHYA (PfrA) acts through heterotrimeric G proteins to stimulate gene expression that results in chloroplast development and anthocyanin biosynthesis (Neuhaus et al., 1993). Three different signal transduction pathways downstream of the G protein were subsequently identified that require cGMP and calcium (Bowler and Chua, 1994; Bowler et al., 1994a). cGMP can stimulate genes such as chalcone synthase (CHS) that are required for anthocyanin biosynthesis, whereas calcium and calcium-activated calmodulin (CaM) can stimulate other genes (e.g. chlorophyll a,b binding protein genes, CAB) necessary for partial chloroplast development. A third pathway, that requires both calcium and cGMP, is utilized to stimulate genes encoding the photosystem I (PSI) and cytochrome b6f (cyt. b6f) complexes (e.g. the gene encoding ferredoxin NADP+ oxidoreductase, FNR). The combination of these three pathways therefore leads to full chloroplast development and anthocyanin biosynthesis. Using similar experiments we wanted, specifically: (i) to address whether PfrA, PrA or both control AS1 regulation, (ii) to determine whether AS1 regulation requires calcium and/or cGMP or whether other signalling molecules are utilized and (iii) to identify specific cis-elements within the AS1 promoter which are targets of PHYA regulation. Our results show that PfrA represses AS1 expression in the light and that it does so via the calcium/cGMP-dependent pathway used to activate other responses, such as FNR gene expression. Hence, probably the same signal transduction pathway is used to simultaneously 'turn on' and 'turn off' different events. One cis-element within the AS1 promoter, which in our assay system displays all the properties of the intact promoter, is highly homologous to the RE1 element within the oat PHYA gene, previously proposed to be a target for phytochrome autoregulation (Bruce et al., 1991). Results AS1–GUS is negatively regulated by PfrA To examine the regulation of AS1 by phytochrome, a plasmid containing 559 bp of the pea AS1 promoter (Tsai, 1991) fused upstream of the gene encoding the reporter β–glucuronidase (AS1–GUS) was injected into subepidermal hypocotyl cells of 7- to 10-day dark-grown wild-type and aurea mutant tomato seedlings. For comparison, equivalent experiments were also performed with a CAB–GUS reporter gene (Neuhaus et al., 1993). Following injection (under green safelight conditions where necessary) the seedlings were exposed to different light irradiations. As we would predict from expression of the endogenous AS1 and CAB genes, AS1–GUS was expressed in injected cells of wild-type seedlings maintained in the dark but not in the light, whereas CAB–GUS was only expressed in the light (Table I). Furthermore, expression of AS1–GUS in the dark could be down-regulated by a pulse of red light, but reactivated by 10 min of far-red irradiation subsequent to the red light pulse. In contrast, CAB–GUS expression could be stimulated in the dark by a pulse of red light and could be down-regulated by a far-red light pulse given immediately after the red light irradiation (Table I). These results thus demonstrate that in wild-type seedlings both AS1–GUS and CAB–GUS expression are regulated by phytochrome, but that this regulation acts in opposite ways, in one case down-regulating and in the other case up-regulating expression. Furthermore, the behaviour of the AS1–GUS gene in these injection experiments clearly reflects endogenous AS1 expression in pea, which has been previously shown to be down-regulated at the level of transcription by white and red light (Tsai and Coruzzi, 1990, 1991). Table 1. Photoregulated expression of AS1–GUS and CAB–GUS in the wild-type and aurea mutant Genotype Reporter gene Light conditions No. injections No. activations Efficiency (%) wt AS1–GUS D 132 12 9.0 wt AS1–GUS L 210 0 wt AS1–GUS R 85 0 wt AS1–GUS R/FR 89 9 10.1 wt CAB–GUS D 79 0 wt CAB–GUS L 132 10 7.6 wt CAB–GUS R 55 6 10.9 wt CAB–GUS R/FR 57 0 au AS1–GUS D 76 8 10.5 au AS1–GUS L 131 13 9.9 au AS1–GUS R 95 7 7.4 au AS1–GUS R/FR 71 8 11.3 au CAB–GUS D 68 0 au CAB–GUS L 122 0 au CAB–GUS R 62 0 au CAB–GUS R/FR 64 0 A summary of AS1–GUS and CAB–GUS expression in response to different light conditions in hypocotyl cells of etiolated wild-type (wt) and aurea (au) seedlings. Following injection of the reporter genes, the seedlings were either transferred to the dark (D) or to white light (L) for 48 h. Seedlings transferred to the dark were injected under green safelight conditions. For phytochrome photoreversibility experiments, seedlings injected under green safelight were irradiated with red light (R) or red light followed by far-red light (R/FR) prior to incubation in the dark for 48 h, as described in Materials and methods. The total number of injections is shown, together with the number of GUS-positive cells observed in each experiment. The efficiency of GUS activation is expressed as a percentage. In injected cells of aurea seedlings both reporter genes were insensitive to the light conditions: AS1–GUS was expressed both in the light and in the dark, whereas CAB–GUS was never expressed (Figure 1 and Table I). The lack of expression in aurea of CAB–GUS, even in the light or after a red light pulse, is consistent with its known requirement for Pfr, because, unlike in the wild-type, etiolated aurea seedlings are largely deficient in phytochrome (Sharma et al., 1993). Furthermore, the fact that in aurea AS1–GUS is expressed under all conditions implies that Pfr normally represses AS1–GUS expression in the light but that in phytochrome-deficient cells it is expressed constitutively. Figure 1.Phenotypes of injected aurea hypocotyl cells after microinjection with signalling intermediates. The AS1–GUS, CAB–GUS, CHS–GUS and FNR–GUS panels show images of cells injected with the reporter genes alone (−) or co-injected with PfrA, GTPγS, calcium, cGMP or calcium plus cGMP. GUS activity was examined as previously described (Neuhaus et al., 1993) following incubation of injected material for 48 h in white light. Images of CAB–GUS, CHS–GUS and FNR–GUS expression patterns are derived from repetitions of previous experiments (Neuhaus et al., 1993; Bowler et al., 1994a). Actual experimental data are shown in Table II. The chloroplasts and anthocyanin panels show representative images of chlorophyll and anthocyanin fluorescence (visualized as described; Neuhaus et al., 1993; Bowler et al., 1994a) observed prior to GUS staining in cells injected with the different signalling intermediates. Chloroplasts generated by PfrA, GTPγS and calcium plus cGMP contain all the photosynthetic machinery, whereas those generated by calcium lack cyt. b6f and PSI (Neuhaus et al., 1993; Bowler et al., 1994a). All images were taken from hand cut sections made through the injected regions of hypocotyls and are derived from independent injections into different seedlings. Approximate intracellular concentrations: PfrA, 20 000 molecules; GTPγS, 50 μM; calcium, 2 μM; cGMP, 50 μM. Arrows indicate the injected cells. Scale bars in bright field micrographs represent 500 μm, those in fluorescent micrographs 10 μm. Download figure Download PowerPoint We have previously found that injection of PHYA into hypocotyl cells of etiolated aurea seedlings in the light can restore chloroplast development and anthocyanin biosynthesis and can activate expression of CAB–GUS, CHS–GUS and FNR–GUS reporter genes (Figure 1 and Table II; Neuhaus et al., 1993; Bowler et al., 1994a). To determine whether PHYA could also regulate AS1–GUS expression, we co-injected AS1–GUS together with PHYA into aurea hypocotyl cells. We found that injection of PfrA (i.e. injection of PHYA in white light conditions) was able to down-regulate AS1–GUS expression in aurea, whereas injection of the Pr form (PrA) (i.e. injection of PHYA in green safelight conditions) could not (Figure 1 and Table II). Furthermore injection of PrA, followed by its conversion in situ to PfrA by a red light pulse could also inhibit expression. This down-regulation by red light could, however, be relieved by subsequent irradiation with far-red light (Table II). These results thus demonstrate that PHYA can control AS1–GUS expression and that it does so in an opposite way compared with CAB–GUS, CHS–GUS and FNR–GUS (Neuhaus et al., 1993; Bowler et al., 1994a). Table 2. Down-regulation of AS1–GUS expression in the aurea mutant by PfrA and its signalling intermediates Co-injected material Light conditions Efficiency (%) (No. activations/No. injections) AS1–GUS CAB–GUS CHS–GUS FNR–GUS C and A D 9.2 (12/130) n.d. n.d. n.d. L 10.0 (37/372) (0/110) (0/113) (0/123) PHYA (20 000) D 7.8 (10/128) n.d. n.d. n.d. PHYA (20 000) L (0/251) 10.8 (11/102) 9.9 (10/101) 8.8 (9/102) C and A PHYA (20 000) R (0/135) n.d. n.d. n.d. PHYA (20 000) R/FR 6.2 (8/129) n.d. n.d. n.d. GTPγS (50 μM) D (0/85) n.d. n.d. n.d. GTPγS (50 μM) L (0/300) 12.4 (13/105) 13.0 (15/115) 11.8 (13/110) C and A GTPγS (50 μM) R (0/72) n.d. n.d. n.d. GTPγS (50 μM) R/FR (0/59) n.d. n.d. n.d. CTX (5000) + GTPγS (1 μM) L (0/129) n.d. n.d. n.d. C and A Ca2+ (2 μM) L 9.3 (20/215) 13.2 (14/106) (0/110) (0/117) C CaM (10 000) L 11.5 (15/131) n.d. n.d. n.d. C cGMP (50 μM) L 9.4 (23/244) (0/113) 13.7 (16/117) (0/109) A Ca2+ (2 μM) + cGMP (50 μM) L (0/296) 8.3 (9/109) 10.0 (11/110) 8.9 (10/113) C and A CaM (10 000) + cGMP (50 μM) L (0/176) n.d. n.d. n.d. C and A CaM (3000) + cGMP (110 μM) L 10.1 (15/148) n.d. n.d. n.d. A CaM (100 000) + cGMP (50 μM) L (0/152) n.d. n.d. n.d. C CaM (10 000) + cGMP (3.5 μM) L (0/152) n.d. n.d. n.d. C PHYA and other compounds were co-injected with AS1–GUS, CAB–GUS, CHS–GUS or FNR–GUS into aurea hypocotyl cells at the concentrations given (expressed as estimated final intracellular concentrations in number of molecules, unless stated otherwise) as described (Neuhaus et al., 1993; Bowler et al., 1994a,b). Efficiency of GUS activation (expressed as %) following different treatments is shown, together with actual experimental data in parantheses (showing the total number of injections and the number of activations). GUS activity was examined 48 h post-injection. Phytochrome photoreversibility experiments were performed as described in the Table I legend and in Materials and methods. For white light experiments, PHYA injections were carried out in white light, whereas for dark, red and far-red experiments, injections were performed under green safelight conditions. Hence, in the former experiments, PHYA was in the PfrA form, while in the latter it was injected in the PrA form. Calmodulin was activated by calcium (CaM) as previously (Neuhaus et al., 1993). A subset of injected cells were examined for chlorophyll (C) and anthocyanin (A) fluorescence in order to confirm previous results (see Figure 1) (Neuhaus et al., 1993; Bowler et al., 1994a,b). In injected cells kept in the dark and in cells treated with red and/or far-red light pulses, no fluorescence was observed (Neuhaus et al., 1993). n.d., not done. Down-regulation of AS1–GUS by PfrA requires calcium and cGMP Previous microinjection experiments in aurea, together with pharmacological studies in soybean SB-P cells, have led to the identification of three major signal transduction pathways used by PfrA to control chloroplast development and anthocyanin biosynthesis (Neuhaus et al., 1993; Bowler and Chua, 1994; Bowler et al., 1994a,b). It was, therefore, of interest to determine whether these pathways are not only used for activation of these responses but also for down-regulation of other responses, e.g. negative regulation of AS1 expression. To test this, we co-injected a range of previously characterized molecules known to stimulate various PfrA responses. Activation of heterotrimeric G proteins, by injection of GTPγS and cholera toxin (CTX), has been shown to stimulate full chloroplast development and anthocyanin biosynthesis in aurea hypocotyl cells (Neuhaus et al., 1993) and to activate the reporter genes CAB–GUS, FNR–GUS and CHS–GUS (Figure 1 and Table II; Bowler et al., 1994a). In contrast, co-injection of GTPγS and CTX with AS1-GUS in aurea led to down-regulation of AS1–GUS and, unlike with PfrA, this response was now unaffected by the light conditions (Figure 1 and Table II). Hence, the response was now light-independent, i.e. it had been uncoupled from the normal stimulus. These data therefore demonstrate that, as for CAB–GUS, FNR–GUS and CHS–GUS activation (Neuhaus et al., 1993; Bowler et al., 1994a), the PfrA-mediated down-regulation of AS1–GUS requires G protein activation and also reveal that there are no light-requiring steps downstream of G protein activation for AS1–GUS down-regulation. This has also been shown for CAB–GUS activation, indicating that the only light-dependent step between PfrA and nuclear gene regulation is likely to be photoreceptor activation (Neuhaus et al., 1993). Injection of calcium and activated calmodulin (CaM) have been found to stimulate CAB–GUS expression and partial chloroplast development in etiolated aurea hypocotyl cells (Figure 1 and Table II; Neuhaus et al., 1993). Conversely, injection of cGMP can stimulate CHS–GUS expression and anthocyanin biosynthesis (Figure 1 and Table II; Bowler et al., 1994a). These molecules therefore control distinct subsets of PfrA responses and act downstream of G protein activation (Neuhaus et al., 1993; Bowler et al., 1994a). To determine if these previously characterized PfrA signalling intermediates also regulate AS1 expression, they were co-injected with AS1–GUS into aurea. Interestingly, neither calcium, activated CaM nor cGMP alone (at concentrations previously found to be effective, 2 μM, 10 000 molecules, and 50 μM, respectively, estimated final intracellular concentrations; Neuhaus et al., 1993; Bowler et al., 1994a,b) could down-regulate AS1–GUS expression in the light in aurea cells (Figure 1 and Table II). However, a combination of calcium or activated CaM together with cGMP was able to effectively block AS1–GUS expression (Figure 1 and Table II), suggesting that the down-regulation of AS1–GUS by PfrA is controlled by the same signalling molecules that it uses to activate other responses. Specifically, it appeared that AS1–GUS down-regulation may be controlled via the same calcium/cGMP-dependent pathway we have found to activate expression of genes encoding PSI and cyt. b6f components, such as FNR (Figure 1 and Table II; Bowler et al., 1994a). PfrA signal transduction pathways have been found to be subject to cross-talk regulation, which has been termed reciprocal control (Bowler et al., 1994b). For example, activity of the calcium/cGMP-dependent pathway has been found to be inhibited by high concentrations of cGMP, but not activated CaM, and to be able to function with significantly lower amounts of cGMP (at least 6-fold) than does the cGMP-dependent pathway. To examine whether regulation of AS1–GUS expression was also modulated by these phenomena, we co-injected different concentrations of activated CaM and cGMP. Indeed, high concentrations of cGMP (110 μM) injected with activated CaM (3000 molecules) were no longer effective in down-regulating AS1–GUS, whereas, in the presence of high concentrations of activated CaM (100 000 molecules) and normal amounts of cGMP (50 μM), down-regulation was still observed, as it was when co-injecting low levels of cGMP (3.5 μM) with activated CaM (10 000 molecules) (Table II). Again, these results indicated that AS1–GUS down-regulation by PHYA was likely mediated by the same calcium/cGMP-dependent pathway that has been previously characterized as activating other responses (Bowler et al., 1994b). It was interesting to observe that in these experiments with PfrA signalling intermediates, phenotypes characteristic of both dark- and light-exposed material were manifested concurrently in the same cell, e.g. although injection of calcium or activated CaM alone in the light resulted in CAB–GUS activation and biogenesis of partially developed chloroplasts and injection of cGMP alone resulted in CHS–GUS activation and anthocyanin pigment biosynthesis, in both cases these cells could not down-regulate AS1–GUS (Figure 1 and Table II). As further evidence that AS1–GUS down-regulation was mediated by the previously characterized calcium/cGMP-dependent pathway, we tested the effect on AS1–GUS expression of previously characterized pharmacological agents. Genistein (an inhibitor of tyrosine and histidine protein kinases; Huang et al., 1992) is known to inhibit the cGMP-dependent pathway, whereas trifluoperazine (a calmodulin antagonist; Massom et al., 1990) and staurosporine (a non-specific protein kinase inhibitor; Rüegg and Burgess, 1989) both inhibit the two calcium-dependent pathways (Bowler et al., 1994b). For these experiments, we injected dark-grown wild-type seedlings and then incubated them in the light in the presence of these different compounds. For comparison, we also examined the expression of CAB–GUS, CHS–GUS and FNR–GUS under the same conditions. As predicted from previous experiments in aurea (Bowler et al., 1994b), CAB–GUS, CHS–GUS and FNR–GUS were expressed in the light in these wild-type seedlings (Table III). Furthermore, as already observed in aurea, CHS–GUS expression was inhibited by genistein, whereas CAB–GUS and FNR–GUS expression were inhibited by trifluoperazine and staurosporine (Table III). These results reveal the consistency of data obtained from aurea and wild-type seedlings. Table 3. Effects of PfrA signal transduction inhibitors on reporter gene expression in wild-type cells Inhibitor Efficiency (%) (No. activations/No. injections) AS1–GUS CAB–GUS CHS–GUS FNR–GUS (0/132) 9.1 (10/110) 9.8 (9/92) 13.5 (12/89) Genistein (0/139) 14.0 (17/121) (0/92) 18.8 (13/69) Trifluoperazine 13.2 (16/121) (0/83) 18.7 (14/75) (0/122) Staurosporine 14.4 (18/125) (0/92) 24.7 (20/81) (0/119) Efficiency of GUS activation (expressed as %) following different treatments is shown, together with actual experimental data in parentheses (showing the total number of injections and the number of activations). Etiolated wild-type seedlings were injected and subsequently incubated for 48 h in white light. Treatment with inhibitors was as described (Bowler et al., 1994b). Concentrations of inhibitors: genistein, 100 μM; trifluoperazine, 200 μM; staurosporine, 60 nM. AS1–GUS, however, was not expressed in the light, as previously observed (Table I), and this down-regulation by light was found to be sensitive to trifluoperazine and staurosporine, but not to genistein (Table III). Based on these data, together with that presented in Table II, it is therefore highly likely that the same signal transduction pathway (i.e. the calcium/cGMP-dependent pathway) is used by PfrA to control both up-regulation of some genes (e.g. FNR–GUS) and down-regulation of others (e.g. AS1–GUS). The target of calcium and cGMP regulation within the AS1 promoter The above data imply that there is a target(s) within the AS1 promoter for PfrA-mediated down-regulation by calcium and cGMP. Most simply, PfrA may act via calcium and cGMP to activate a repressor that binds to such a sequence. To date, the best characterized cis-acting element found to be important for phytochrome-mediated down-regulation is RE1, an 11 bp GC-rich sequence centered at −75 bp within the oat PHYA promoter (Bruce et al., 1991). When the RE1 sequence is mutated by linker scanning mutagenesis, this promoter retains maximal expression following a far-red light pulse but is no longer down-regulated by a red light pulse (Bruce et al., 1991). Interestingly, the RE1 core sequence, TGGG, is present within other PHYA promoters and can also be found in the promoters of all genes so far characterized as being down-regulated by light (Figure 2). Examination of the AS1 promoter sequence revealed the presence of two such sequences, albeit on the opposite DNA strand with respect to monocotyledon PHYA promoters, showing significant homology with the RE1 core sequence, one centered at −43 and the other centered at −160 (Figure 2). Thus, it appeared possible that these elements may be the targets for PfrA-mediated repression within the AS1 promoter. To determine whether these sequences were required for down-regulation of AS1–GUS by light, we performed competition experiments using a tetramer of the most proximal RE1-related element within the AS1 promoter (denoted RE3, centered at −43) (Figure 2). Similar competition experiments have recently been performed in tobacco cotyledon cells to study regulation of the cauliflower mosaic virus (CaMV) −90 35S promoter (Neuhaus et al., 1994). Figure 2.RE1-related elements within PHYA promoters and the pea AS1 promoter. Sequences were derived from the following sources: oat PHYA, Hershey et al., 1985; rice PHYA, Kay et al.,