Planta

Planta (1989) 177:228 236

9 Springer-Verlag 1989

A stable blue-light-derived signal modulates ultraviolet-light-induced activation of the chalcone-synthase gene in cultured parsley cells S. OhP, K. Hahlbrock 2, and E. SchMer 1 1 Institut ffir Biologie II/Botanik der Universitfit Freiburg, Schfinzlestrasse 1, D-7800 Freiburg i.Br., and 2 Max-Plank-Institut ffir Zfichtungsforschung, Abteilung Biochemie, Egelspfad, D-5000 K61n 30, Federal Republic of Germany

Abstract. Run-off transcription assays were used to demonstrate that both the ultraviolet (UV)-B and blue-light receptors control transcription rates for chalcone-synthase m R N A in the course of light-induced flavonoid synthesis in parsley (Petroselinum crispum Miller (A.W. Hill)) cell-suspension cultures. Blue and red light alone, presumably acting via a blue-light receptor and active phytochrome (far-red absorbing form) respectively, can induce accumulation of chalcone-synthase m R N A . The extent of the response is however considerably smaller than that obtained when these wavebands are applied in combination with UV light. A preirradiation with blue light strongly increases the response to a subsequent UV pulse and this modulating effect of blue light is stable for at least 20 h. The modulating effect is abolished by a UV induction but can be reestablished by a second irradiation with blue light.

Key words: Blue light - Cell culture (gene regulation) - Chalcone synthase - m R N A transcription - Petroselinum (cell culture) Photoreceptor - Signal transduction chain Transcription (regulation)

Introduction

Flavonoid synthesis in parsley (Petroselinum crispum) cell-suspension cultures is regulated by light in a complex manner (Wellmann 1975a; Hahlbrock 1976; Ebel and Hahlbrock 1977). The metabolic endproducts, flavone and flavonol glycosides, probably function as ultraviolet (UV) protectants in intact plants (Caldwell 1971; Wellmann 1983; Schmelzer et al. 1988). Abbreviations: Pfr=far-red-absorbing form of phytochrome;

UV = ultraviolet

Chappell and Hahlbrock (1984) showed that the induction of flavonoid synthesis by a UV-Bcontaining white-light source is preceded by the activation of genes encoding two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, and the key enzyme of flavonoid metabolism, chalcone synthase. Three operationally defined photoreceptors are involved in the regulation of flavonoid accumulation in this system, a UV-B-light receptor, a bluelight receptor (cryptochrome) and phytochrome. While UV light is essential for effective induction, blue light and red/far-red light, the latter acting via phytochrome, modulate the response (DuellPfaff and Wellmann 1982). Irradiation with blue prior to UV light results in an earlier increase in and higher maximum levels of chalcone-synthase m R N A (Bruns et al. 1986). Ultraviolet light itself establishes a rather high percentage of the active far-red-absorbing form of phytochrome (75 % Pffr, Duell-Pfaff and Wellmann 1982). Irradiation with far red light results in a partial reversibility of the UV-light-induced accumulation of flavonoids (Duell-Pfaff and Wellmann 1982) as well as a shorter transient increase in chalcone-synthase m R N A (Bruns et al. 1986). Chalcone synthase is particularly suitable for studies of the interaction of different photoreceptors on gene regulation in parsley cell cultures, for several reasons. In these cultures, the flavonoid pathway, including chalcone synthase, by far the most abundant of its enzymes, is selectively and very efficiently induced by light. It is insensitive to other environmental stimuli such as fungal elicitor, infection, heat shock and other types of stress (Hahlbrock et al. 1981). The genomic background of chalcone synthase in parsley is well characterized. Only one gene encodes chalcone synthase. It occurs in two allelic forms. Allele 1 contains

S. Ohl et al. : Modulation of UV-light induced-chalcone-synthase-gene activation

a 927-bp transposon-like insertion at position - 538, relative to the first of two transcription start sites. Cell cultures homozygous with respect to chalcone synthase were used to demonstrate that both alleles are responsive to UV light (Herrmann et al. 1988). The measurement of changes in the amount or activity of m R N A does not differentiate between alterations in m R N A synthesis and degradation. It was therefore our aim to investigate whether or not all three photoreceptors act at the transcriptional level, thus enabling us to analyse the interaction of the different photoreceptors further. The newly established parsley cell cultures (Schmelzer et al. 1988; Herrmann et al. 1988) used for the present analysis differed in some details from the cell culture studied previously (Bruns et al. 1986), particularly with regard to the magnitude of the red-light effect. However, the UV and blue-light effects were qualitatively the same. We now present evidence for a stable blue-light-derived signal interacting with the UV-light-triggered signal-transduction chain. Moreover, blue light alone is shown to induce chalcone-synthase m R N A to a low but significant level. The two effects of blue light can be distinguished by certain criteria. Material

and methods

Plant material. Cell-suspension cultures of parsley (Petroselinum crispurn Miller (A.W. Hill)), either heterozygous or homozygous for each of two chalcone-synthase alleles (horn I and horn 2) were maintained in the dark and subcultured every 7 d as described previously (Kombrink and Hahlbrock 1986). For irradiation, cells of 6-d-old cultures were collected aseptically on porous glass filters, transferred to fresh medium at a concentration of 6 g cells to 75 ml medium and kept in the dark for another 24 h before U V irradiation. Aliquots of 6 g cells were irradiated in glass vessels covered with UV-transmitting plastic lids, harvested as described (Hahlbrock 1976) and stored at --70 ~ C. All manipulations of the cells prior to irradiation were performed under a green safelight (Mohr and A p p u h n 1963).

Light sources. Pbilips (Hamburg, F R G ) TL 40W/12 tubes (2m~x=310 nm) were used as the UV light source. Based on the spectral fluence-rate distribution published by Catdwell et al. (1986), the fluence rate at a distance of 30 cm from a single fluorescent tube was calculated as 4.4 W . m -2. In our experiments, 5-6 tubes were used resulting in fluence rates of 21 and 3 2 W . m -2. Blue light (2max=436 rim, 4.8 W . m -2) and red light (2m. x = 658 nm, 6.8 W . m -z) were obtained from standard sources (Mohr et al. 1964; Hanke et al. 1969). Long-wavelength far-red light was obtained from a Leitz Prado projector combined with a far-red glass filter (Schott R G 9, 5 ram, 2max=775 rim, 5.5 W- m - z ; Schott, Mainz, FRG). In-vitro transcription. An aliquot of 6 g of cells was ground to a fine powder in liquid nitrogen, thawed in 160 ml extraction

229

buffer (0.25 M sucrose, 10 m M NaC1, 10 m M N-morpholinoethanesulphonic acid, pH 6,5 m M ethylenediaminetetraacetic acid (EDTA), 0.15 m M spermine, 0.5 m M spermidine, 20 m M mercaptoethanol, 0.1% bovine serum albumin (BSA), 0.6% Triton X-100 (octylphenoxy polyethoxyethanol) and 2 % Dextran T 40), and filtered through two nylon sieves of mesh size 80 and 20 gin, respectively. The filtrate was further purified and concentrated by three successive centrifugations (1000.g, 5 min). Each pellet was resuspended in extraction buffer and recentrifuged. The final pellet, containing an enriched nuclear fraction, was resuspended in extraction buffer in which the Dextran had been replaced by 20% glycerol. All steps were carried out at 4 ~ C. For quantification, nuclei were counted in a counting chamber (0.2.0.0165 m m 2) after staining with methyl green pyronine (Jensen 1962). The method used for in-vitro transcription was as described by M6singer et al. (1985), except that 2.107 nuclei were used per assay. Extraction of nuclear R N A was carried out according to M6singer et al. (1985) with some minor modifications. After the second chloroform extraction, the aqueous phase was applied to a Sephadex G 50 spin column (packed bed volume 5 ml, equilibrated in STE buffer according to Maniatis (1982)) and centrifuged at 1600.g for 4 rain. One volume of 1.3 M NaC1 was added to the eluate and the solution was recentrifuged at 5000-g for 20 min. The supernatant was precipitated with ethanol and the resulting pellet dried and resuspended in 1 ml hybridization buffer. To determine the total incorporation rate, aliquots were taken as described by M6singer and Sch/ifer (1984).

Extraction of total RNA. Total R N A was isolated as described by Langridge et al. (1982). Final D N A contamination was removed by selectively pelleting R N A in 2 M LiC1 (Pelham 1985). R N A was stored at - 7 0 ~ C. Concentration and purity were determined spectrophotometrically by measuring the absorbance at 260 nm and the ratio A 2 6 o / A 2 8 o.

Dot-blot hybridization. Recombinant plasmids containing sequences specific for parsley 4-coumarate:CoA Iigase (pc4CL) and chalcone synthase (pcCHS) (Kuhn et al. 1984) were prepared according to Birnboim and Doly (1979). Insert D N A was isolated by Pst I (chalcone synthase) or EcoR I digestion (4-coumarate:CoA ligase) of plasmids and subsequent separation either on an agarose gel (Dretzen et al. 1981) or using a " B i o t r a p " (Schleicher u. Schuell, Dassel, F R G ; method described by G6bel et al. 1987). Plasmid D N A containing 400 ng insert D N A was dotted onto nitrocellulose strips (Parnes et al. 1981). Blots were prehybridized for at least 6 h at 42 ~ C in hybridization buffer (50% formamide, 5 x SSC (1 x SSC=0.15 M NaC1, 0.015 M sodium citrate), 50 m M sodium phosphate, pH 6.5, 0.2% sodium dodecylsulfate (SDS), 5 x D e n h a r d t ' s solution (Denhardt 1966), 100 gg-ml-1 each t R N A and polyadenylated RNA). Hybridization was carried out for 36 h at 42 ~ C in the buffer containing 1.5.107-3.0 - 107 cpm of 32p-labelled R N A in a volume of I ml. Filters were washed as described previously (Bruns et al. 1986), air-dried and exposed to X-ray film at - 8 0 ~ C using intensifying screens. For quantification, dots were cut out of the nitrocellulose and filter-bound radioactivity was counted in a liquid scintillation counter. Values are given relative to the total incorporated counts. No background hybridization was detected against pBR322. Curves were fitted by eye. For Northern blots, 10 pg R N A was denatured in 50% formamide, 6% formaldehyde, I x Mops-buffer ( = 2 0 m M Nmorpholinopropanesulphonic acid pH 7.0, 5 m M sodium acetate, 1 m M EDTA) at 65~ for 5 rain and separated on an agarose-formaldehyde gel (1.25% agarose in I x Mops-buffer

230

S. OhI et al. : Modulation of UV-light induced-chalcone-synthase-gene activation

containing 6% formaldehyde). The R N A was transferred in 20 x SSC onto nitrocellulose and baked at 80 ~ C for 2 h. For preparation of dot-blots, a dilution series of 0.3-5.0 gg denatured R N A was dotted onto nitrocellulose as described by Bruns et al. (1986). Insert D N A (0.1 gg) was labelled by nick translation with 1.9.106 Bq [32p]cytidine 5'triphosphate (1.1. l0 s B q . m o l - 1) and used as a hybridization probe. Hybridization and washing of Northern and dot-blot filters was performed as described by Bruns et al. (1986). For quantification, dots were cut out and filter-bound radioactivity counted in a liquid scintillation counter.

Results and discussion

Ultraviolet-B and blue receptors are both effective at the transcriptional level. The parsley cell culture used in this investigation was established only recently. It is still diploid (D. Scheel, Max-PlanckInstitut ffir Zfichtungsforschung, K61n, FRG, personel communication) and of an origin different from that used in previous investigations. This might explain quantitative and qualitative differences between our results and previously reported findings. Parsley cells were either irradiated with 5 min UV light (32 W. m - z ) alone or with 5 h blue light (4.8 W - m -2) followed by 5 min UV light. Cells were then transferred to the dark and harvested at various times after the light treatment. Nuclei were isolated and in-vitro transcription rates determined by measuring the incorporation of [3Zp] uridine 5'-triphosphate (UTP) into those in-vivo transcripts which hybridized to excess nitrocellulosebound chalcone-synthase copy D N A (cDNA). 20

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Fig. 1. In-vitro transcription kinetics obtained for chalcone synthase. Parsley cells were either irradiated with 5 min U V light (32 W - m - Z ; D) or received an additional 5 h blue irradiation prior to U V (zx). The data are mean values of three independent experiments

Figure 1 shows a comparison of the kinetics of chalcone-synthase transcription obtained for UV and blue + UV irradiations. The transcription maximum induced by a UV-light pulse alone occurred about 3-4 h after induction. This is at least 2 h earlier than the chalcone-synthase transcription maximum observed by Chappell and Hahlbrock (1984) in the cell culture used previously. This difference is probably caused by the difference in the cell cultures and by the use of a different UV-light source. The action of a separate blue-light receptor in addition to the UV receptor is clearly seen at the transcriptional level. A blue preirradiation results in an earlier increase in transcription rates and a higher maximal induction. A rather high transcription rate of about 2.5 ppm was measured in darkgrown cells. This high level is most probably the consequence of background hybridization in the in-vitro transcription assay, since no chalcone-synthase m R N A could be detected in Northern blots (Fig. 2 and inset Fig. 9). Blue light alone induces chalcone synthase. Many previous publications have drawn attention to the obligatory role of UV light for light induction of genes involved in flavonoid metabolism in parsley cell-suspension cultures. Blue or red light alone showed no effect. In our cell culture, however, we could demonstrate that a low but significant amount of chalcone-synthase m R N A accumulates under constant blue light. Figure 2 shows a comparison of the maximal m R N A levels induced by blue light and by the blue + U V irradiation. Blue light induces about 10% of the m R N A level accumulating after a blue + UV irradiation. This effect could be demonstrated in three separate experiments and was caused solely by an increase in the chalcone-synthase-specific hybridization signal. The time courses for the accumulation of chalcone-synthase m R N A in constant blue light and its subsequent disappearance on transferring the cells to the dark is shown in Fig. 3. In blue light, the m R N A amount increases for up to about 4 h, after which a constant level is maintained for at least 20 h. If the cells are transferred back to darkness, the m R N A declines to a low background level within a few hours. This effect of blue light must be ascribed to the blue-light receptor. Contamination of the blue light source by UV light was excluded by the use of a 400-rim cut-off filter in a control experiment (data not shown). The published action spectra for UV-B receptors in several plants (Wellmann 1975b; Yatsuhashi et al. 1982) indicate that the

S. Ohl et al. : Modulation of UV-light induced-chalcone-synthase-gene activation 120

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Time after onset of blue [h] Fig. 3. Kinetics of the accumulation of chalcone-synthase m R N A under constant blue light (D) and following transfer of the cells back to darkness 12 h after the onset of blue light (arrow, zx). The data are mean values of two to three independent experiments normalized to the blue-light-induced maxim u m levels

Fig. 2. Northern blot of chalcone-synthase (CHS) m R N A accumulation induced in suspension-cultured parsley cells by different light treatments: D = dark control ; B = 8 h blue light; UV= 2 rain UV light (21 W - m z); B-UV=5 h blue followed by 2 rain U V light. Cells were harvested directly (B) or 4 h after the end of irradiation (UV, B-UV)

cross-section of a UV-B receptor in the blue waveband (above 400 rim) would be reduced by a factor of at least 500. The possibility that the blue-lightmediated effect was the result of absorption by the UV-B receptor in the blue spectral range can thus also be excluded. Furthermore, the blue-light-mediated induction of chalcone-synthase m R N A differs qualitatively from UV induction. Under constant blue light, a certain constant m R N A level is maintained for many hours, whereas all available data indicate that the UV-light-induced m R N A increase is transient even under constant irradiation (Schmelzer et al. 1988) and irrespective of the UV fluence (Bruns et al. 1986). The independent blue-light-mediated increase in chalcone-synthase m R N A described here cannot account for the modulating effect of blue light on the UV-induced response. It is too weak to explain the modulation in terms of a simple additive effect,

and the modulating effect of blue light is stable and can be observed even 20 h after irradiation with blue light (see below). The level of m R N A induced by blue light alone, on the other hand, declines rapidly after the cells are transferred back to the dark.

Fluence-response relationship for blue light. Since it could be shown that UV light and blue light both induce chalcone synthase by increasing the transcription rates of the corresponding gene, a more detailed study of the coaction(s) between UV light and blue light was carried out by measuring the amounts of chalcone-synthase m R N A by means of Northern and dot-blot hybridization of total R N A to the corresponding cDNA. The fluence requirements of the blue-light receptor were determined by irradiating cells for 0-8 h with blue light, transferring them to darkness for 1 h and then inducing them with a subsaturating UV pulse (2 min, 21 W . m - 2 ) . The transcription and m R N A kinetics obtained after UV and blue + UV irradiation (Figs. 1, 8) indicated that the strongest effect of blue light was to be expected during the initial period of m R N A increase. Accumulation of m R N A was thus measured 2 and 4 h after UV irradiation. Figure 4 shows the amount of m R N A plotted against the duration of the blue preirradiation. Increasing the blue-light fluence resulted in an increase in the levels of chalcone-synthase m R N A both 2 and 4 h

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S. Oh1 et aL : Modulation of UV-light induced-chalcone-synthase-gene activation

after UV irradiation. The effect was saturated by blue-irradiation periods longer than 2 h. The values measured 4 h after light treatment might indicate an optimum for 2 h blue irradiation with slightly decreasing responses at higher blue-light fluences.

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duration of blue irradiation [h] Fig. 4. Fluence-response relationship for the blue-light-mediated effect on chalcone-synthase-mRNA induction by UV light. Parsley cells were irradiated with blue light for up to 8 h and induced by a brief UV light pulse (2 min, 21 W - m -2) 1 h after the end of the blue irradiation. The amount of chalcone-synthase m R N A found 2 h (D) and 4 h (A) after UV induction was determined. Horizontal lines indicate the m R N A levels induced by UV light alone. The data are mean values of three to four independent experiments normalized to the m R N A maximum levels 4 h after induction by b l u e + U V light. (SEs are only given for four values)

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the UV-induced m R N A accumulation of chalcone synthase. To determine the half-life of this effect, the cells were given a subsaturating blue irradiation (1 h) followed by varying dark periods before an inducing UV-light pulse. Figure 5 shows the amounts of chalcone-synthase m R N A which had accumulated 2 h and 4 h after the UV-light pulse, plotted against the length of the dark period between the blue and the UV-light treatments. The blue-light-mediated rise in transcript level is maintained even if the dark period is as long as 19 h (e.g. 20 h from onset of blue light). Consequently, a blue-light-derived signal must be very stable (half-life of the order of at least 1 2 d). A period of 1-2 h between the blue and UV irradiaton is sufficient for full expression of the blue-light effect. Thus the time required for transduction of the blue-light signal must be of the order of 2 h. It was not possible to determine the minimal time necessary for signal transduction more precisely, because this required the use of shorter periods of blue irradiation, resulting in smaller and less appreciable responses. It was then investigated whether such a stable blue-light-derived signal is necessary for induction by UV light and whether it can also be produced by UV light acting via the UV-B receptor. The problem was approached by means of a double UV-induction irradiation programme. It was necessary to leave at least 14 h between two UV inductions in order to decrease the amount of m R N A induced by the first pulse to a sufficiently low level for an accurate measurement of the second induction response. The accumulation of chalcone-synthase following a second UV-light pulse which was given 14 h after the first is shown in Fig. 6. No appreciable m R N A increase was observed during the first 12 h after induction. The m R N A level then increased to reach a maximum about 4 h after UV irradiation. This maximum occurred about 3 h earlier than the maximum induced by the first UV pulse (compare Fig. 8), and might have been the result of some kind of adaptation effect. Nevertheless, the apparent lag phase of 1-2 h is typical for a UV induction without blue preirra-

S. Oh1 et al. : M o d u l a t i o n o f U V q i g h t i n d u c e d - c h a l c o n e - s y n t h a s e - g e n e activation

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diation. Thus, apparently, the first UV-light pulse did not have a "blue effect" (accelerated m R N A increase) on the second induction, even though the effect of blue light can be stored for times much longer than 14 h (compare Fig. 4). The results thus indicate that either UV light does not produce a signal equivalent to the bluelight signal or that signal is used up or modified by some event involved in UV light induction. To test whether the blue-light-mediated signal is modified during induction by UV light, cells were given 3 h blue light prior to the first UV-light pulse and the m R N A accumulation was measured after a second UV pulse applied 14 h after the first (Fig. 6). No "blue effect" could be observed, both kinetics are identical within experimental error. To ensure that blue light is, in principal, able to stimulate UV-light-induced m R N A accumulation for a second time the effect of blue light on the second UV-light induction was also tested. In this experiment, a rather high m R N A level (equivalent to 50% relative m R N A amount in Fig. 6, data not shown) was measured at the beginning of the kinetic (14 h after the first UV-light pulse). This higher level was probably the consequence of a coaction between the blue light and the first UV-light pulse resulting in a raising of the level of chalcone-synthase m R N A during blue irradiation. Such an effect of blue light given after UVlight induction was reported by Duell-Pfaff and Wellmann (1982). The fact that blue light still can act after UV treatment is of interest with respect to its mechanism of action, but was not further investigated in this paper. To circumvent the interaction between blue light and a UV pulse given previously, the time between first and second UV pulses was increased

to 20 h. The irradiation program was thus, 3 h blue light, UV1, 17 h dark, 3 h blue light, UV2. In a control experiment the second blue irradiation was omitted. As shown in Fig. 6, the initial m R N A level declined further 20 h after the first induction and the second blue irradiation no longer resulted in a substantially higher m R N A level. The effect of the second blue irradiation on the UV induction is, however, clearly demonstrated by a comparison of the two kinetics. The second blue irradiation again eliminates the delay in the response to UV light. This result thus allows one to conclude that blue light results in the production of a stable signal, which is modified by a step involved in UVlight induction. This signal can be induced again by a second blue irradiation. Since UV light modifies the signal, it is not possible to determine whether UV light also results in production of the signal.

Both chalcone-synthase alleles respond to UV and blue light. In the parsley cell culture, there is only one gene coding for chalcone synthase. This gene occurs in two allelic forms, the only major difference being that one form possesses a transposonlike insertion in the putative regulatory region upstream of the transcription start site (Herrmann et al. 1988). The possibility thus exists that the two chalcone-synthase alleles might respond differently to light treatments. For this reason, we tested the responses to blue, UV and b l u e + U V irradiation of two cell cultures from parsley plants homozygous for the two chalcone-synthase alleles. As shown in Fig. 7, both cell cultures (hom 1 and horn 2) respond almost identically to all three light treatments. Thus, the transposon like insertion

234

S. Ohl et al. : Modulation of UV-light induced-chalcone-synthase-gene activation 125

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found in allele 1 and its surrounding sequences are probably not involved in light regulation.

Effect of red light. In order to investigate whether phytochrome is also involved in gene regulation in this system, cells were given a J0-min far-red light pulse immediately after the UV irradiation so as to revert almost all Pfr back to the inactive red-adsorbing form, Pr (0.01% Pfr). No appreciable differences between the kinetics for UV plus far-red and UV alone could be found on measuring either run-off transcription or m R N A accumulation (Fig. 8). Thus, in this cell culture, chalcone synthase does not appear to respond to a far-red light treatment after UV light. Either the gene does not respond to phytochrome at all or the effect of phytochrome is already saturated at the very low level of Pfr established by far-red light, or the coupling time of the phytochrome response is too fast to allow reversion after 5 min of UV light. Spectroscopic measurements showed that the phytochrome content of this cell culture was equivalent to 8.5-10 -4 A(AA), which is comparable to the values obtained for the parsley cell culture used previously (Gottmann and Schfifer 1982). We therefore tested whether irradiation with red light prior to UV treatment has an effect on the UVlight-induced accumulation of chalcone-synthase

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Time after onset of UV [h] Fig. 8. Effect of far-red light on the in-vitro transcription rate of UV-induced chalcone synthase (n, zx) and on the kinetics of m R N A accumulation (O, v). Suspension-cultured parsley cells were either irradiated with 5 min UV light (32 W . m -2) alone (n, O) or received an additional 10-min far-red pulse immediately after UV (zx, v). The data are mean values of three (n, zx) and two (O, v) independent experiments normalized to the corresponding UV-induced maxima

mRNA. Duell-Pfaff and Wellmann (1982) reported that a red preirradiation resulted in increased flavonoid accumulation, although to a smaller extent than that induced by a blue preirradiation. The cells received a 5-h red irradiation followed by a brief UV light pulse. In Fig. 9 the subsequent accumulation of chalcone-synthase-mRNA is compared with that of the UV control. The data show a small but significant red-light-mediated increase in chalcone-synthase mRNA. Thus phytochrome does appear to be involved in light regulation of chalcone synthase. The effect of blue light cannot be a consequence of the action of phytochrome alone, since the bluelight-mediated effect is much greater than that mediated by red light, whereas the photoconversion cross-section of phytochrome is much higher in red than in blue light. A prolonged irradiation with red light resulted in a slight increase in chalconesynthase mRNA. If the autoradiogram of a Northern blot is strongly overexposed a weak red-lightinduced signal appears, which is absent in the dark control (inset Fig. 9). Conclusion

The results described in this paper show that red, blue and UV light given alone all lead to a measur-

S. Ohl et al. : Modulation of UV-light induced-chalcone-synthase-gene activation

235

Fig. 9. Effect of red light on the accumulation of chalconesynthase m R N A induced by UV light. Parsley cells received either 2 rain UV (21 W . m -2) alone (n) or an additional 5 h red light prior to the UV pulse (zx). The data are mean values of three independent experiments normalized to the UV-induced highest values. Inset: Northern blot of chalcone-synthase m R N A from dark grown cells (D) and cells which had received either 8 h blue light (B) or 4 h red light (HR). 26 S and 18 S r R N A serve as size markers

able accumulation of chalcone-synthase m R N A in parsley cell cultures. Higher levels of expression, however, require activation of a UV-B photoreceptor. The effect of red light given alone shows that the presence o f Pfr can result in accumulation of chalcone-synthase m R N A without the necessity for simultaneous activation of a blue or UV-B photoreceptor. Whether these latter two photoreceptors can induce chalcone-synthase-mRNA accumulation without Pfr being present cannot be determined using the methods applied in this paper. Run-off transcription assays showed that both the blue-light receptor working in combination with a UV irradiation and the UV-B receptor act at the level of transcription. Owing to the low levels of expression obtained with red or blue light alone, it was not possible to demonstrate whether Pfr and the blue-light receptor also act at this level. This must, however, be considered to be very probable. Kinetic studies of blue-light-mediated responses demonstrated the existence of two separate effects of blue light. (i) Under continuous irradiation with blue light an increased constant level of

chalcone-synthase m R N A was observed, decreasing rapidly after transition to darkness. (ii) A very stable blue-light-mediated modification accelerates the chalcone-synthase-mRNA accumulation which occurs after irradiation with UV light. This stable modification is nullified after a UV irradiation but can be re-established by a second irradiation with blue light. 4-Coumarate:CoA ligase showed a pattern of light regulation very similar to that for chalcone synthase (data not shown). This indicates that the coactions of the three photoreceptors described here may be general to the enzymes of flavonoid metabolism in parsley. We thank Martina Krenz for preparing plasmid and insert D N A and Renate Wirtz for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie.

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